Prevention of disulfide bond reduction during recombinant production of polypeptides

ABSTRACT

The invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/043,758, filed Oct. 1, 2013, which is a divisional of U.S.application Ser. No. 13/354,223, filed Jan. 19, 2012, now U.S. Pat. No.8,574,869, issued Nov. 5, 2013, which is a continuation of U.S.application Ser. No. 12/217,745, filed Jul. 8, 2008, now abandoned,which is a non-provisional application filed under 37 CFR 1.53(b)(1),claiming priority under 35 USC 119(e) to provisional application No.60/948,677, filed Jul. 9, 2007, the contents of which applications areherein incorporated by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392013903SeqList.txt,date recorded: Apr. 6, 2017, size 67 KB).

FIELD OF THE INVENTION

The invention concerns methods and means for preventing the reduction ofdisulfide bonds during the recombinant production ofdisulfide-containing polypeptides. In particular, the invention concernsthe prevention of disulfide bond reduction during harvesting ofdisulfide-containing polypeptides, including antibodies, fromrecombinant host cell cultures.

BACKGROUND OF THE INVENTION

In the biotechnology industry, pharmaceutical applications require avariety of proteins produced using recombinant DNA techniques.Generally, recombinant proteins are produced by cell culture, usingeither eukaryotic cells, such as mammalian cells, or prokaryotic cells,such as bacterial cells, engineered to produce the protein of interestby insertion of a recombinant plasmid containing the nucleic acidencoding the desired protein. For a protein to remain biologicallyactive, the conformation of the protein, including its tertiarystructure, must be maintained during its purification and isolation, andthe protein's multiple functional groups must be protected fromdegradation.

Mammalian cells have become the dominant system for the production ofmammalian proteins for clinical applications, primarily due to theirability to produce properly folded and assembled heterologous proteins,and their capacity for post-translational modifications. Chinese hamsterovary (CHO) cells, and cell lines obtained from various other mammaliansources, such as, for example, mouse myeloma (NS0), baby hamster kidney(BHK), human embryonic kidney (HEK-293) and human retinal cells, such asthe PER.C6® cell line isolated from a human retinal cell, which provideshuman glycosylation characteristics, and is able to naturally produceantibodies that match human physiology, have been approved by regulatoryagencies for the production of biopharmaceutical products.

Usually, to begin the production cycle, a small number of transformedrecombinant host cells are allowed to grow in culture for several days(see, e.g., FIG. 23). Once the cells have undergone several rounds ofreplication, they are transferred to a larger container where they areprepared to undergo fermentation. The media in which the cells are grownand the levels of oxygen, nitrogen and carbon dioxide that exist duringthe production cycle may have a significant impact on the productionprocess. Growth parameters are determined specifically for each cellline and these parameters are measured frequently to assure optimalgrowth and production conditions.

When the cells grow to sufficient numbers, they are transferred tolarge-scale production tanks and grown for a longer period of time. Atthis point in the process, the recombinant protein can be harvested.Typically, the cells are engineered to secrete the polypeptide into thecell culture media, so the first step in the purification process is toseparate the cells from the media. Typically, harvesting includescentrifugation and filtration to produce a Harvested Cell Culture Fluid(HCCF). The media is then subjected to several additional purificationsteps that remove any cellular debris, unwanted proteins, salts,minerals or other undesirable elements. At the end of the purificationprocess, the recombinant protein is highly pure and is suitable forhuman therapeutic use.

Although this process has been the subject of much study andimprovements over the past several decades, the production ofrecombinant proteins is still not without difficulties. Thus, forexample, during the recombinant production of polypeptides comprisingdisulfide bonds, especially multi-chain polypeptides comprisinginter-chain disulfide bonds such as antibodies, it is essential toprotect and retain the disulfide bonds throughout the manufacturing,recovery and purification process, in order to produce properly foldedpolypeptides with the requisite biological activity.

SUMMARY OF THE INVENTION

The instant invention generally relates to a method for preventingreduction of a disulfide bond in a polypeptide expressed in arecombinant host cell, comprising supplementing the pre-harvest orharvested culture fluid of the recombinant host cell with an inhibitorof thioredoxin or a thioredoxin-like protein.

In one embodiment, the thioredoxin inhibitor is added to the pre-harvestculture fluid.

In another embodiment, the thioredoxin inhibitor is added to theharvested culture fluid.

In a further embodiment, the thioredoxin inhibitor is a direct inhibitorof thioredoxin.

In all embodiments, the thioredoxin inhibitor may, for example, be analkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative.

In a further embodiment, the thioredoxin inhibitor is a specificinhibitor of thioredoxin reductase.

In a still further embodiment, the thioredoxin inhibitor is a goldcomplex, where the gold complex may, for example, be aurothioglucose(ATG) or aurothiomalate (ATM). While the effective inhibitoryconcentration may vary, it typically is between about 0.1 mM and 1 mM.Similarly, the minimum effective inhibitory concentration variesdepending on the nature of the polypeptide and overall circumstances,and is typically reached when the ATG or ATG concentration is at leastabout four-times of thioreduxin concentration in the pre-harvest orharvested culture fluid.

In another embodiment of this aspect of the invention, the thioredoxininhibitor is a metal ion, where the metal ion, without limitation, maybe selected from the group consisting of Hg²⁺, Cu²⁺, Zn²⁺, Co²⁺, andMn²⁺. When the metal ion is added in the form of cupric sulfate, theeffective inhibitory concentration generally is between about 5 μM andabout 100 μM, or between about 10 μM and about 80 μM, or between about15 μM and about 50 μM. The minimum inhibitory concentration of cupricsulfate also varies, but typically is reached when cupric sulfate isadded at a concentration at least about two-times of thioredoxinconcentration in the pre-harves or harvested culture fluid.

In different embodiment, the thioredoxin inhibitor is an oxidizingagent, e.g., an inhibitor of G6PD, such as, for example, pyridoxal5′-phosphate, 1 fluoro-2,4 dinitrobenzene, dehydroepiandrosterone (DHEA)or epiandrosterone (EA); cystine or cysteine. Typical effectiveinhibitor concentrations of DHEA are between about 0.05 mM and about 5mM, or between about 0.1 mM and about 2.5 mM.

In a further embodiment, the thioredoxin inhibitor is an inhibitor ofhexokinase activity, including, without limitation, chelators of metalions, such as, for example, ethylenediamine tetraacetic acid (EDTA).EDTA is typically added and effective at a concentration between about 5mM and about 60 mM, or about 10 mM and about 50 mM, or about 20 mM andabout 40 mM.

In other preferred embodiments, the inhibitor of hexokinase activity isselected from the group consisting of sorbose-1-phosphate,polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose,xylose, and lyxose.

Other inhibitors include cystine, cysteine, and oxidized glutathionewhich are typically added at a concentration at least about 40-times ofthe concentration of the polypeptide in question in the pre-harvest orharvested culture fluid.

In a still further embodiment, the thioredoxin inhibitor is an siRNA, anantisense nucleotide, or an antibody specifically binding to athioredoxin reductase.

In another embodiment, the thioredoxin inhibitor is a measure indirectlyresulting in the inhibition of thioredoxin activity. This embodimentincludes, for example, air sparging the harvested culture fluid of therecombinant host cell, and/or lowering the pH of the harvested culturefluid of the recombinant host cell.

In various embodiments, indirect means for inhibiting thioredoxinactivity, such as air sparging and/or lowering of the pH, can becombined with the use of direct thioredoxin inhibitors, such as thoselisted above.

In all embodiments, the polypeptide may, for example, be an antibody, ora biologically functional fragment of an antibody. Representativeantibody fragments include Fab, Fab′, F(ab′), scFv, (scFv)₂, dAb,complementarity determining region (CDR) fragments, linear antibodies,single-chain antibody molecules, minibodies, diabodies, andmultispecific antibodies formed from antibody fragments.

Therapeutic antibodies include, without limitation, anti-HER2 antibodiesanti-CD20 antibodies; anti-IL-8 antibodies; anti-VEGF antibodies;anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies;anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-Tissue Factor(TF) antibodies; anti-human α₄β₇ integrin antibodies; anti-EGFRantibodies; anti-CD3 antibodies; anti-CD25 antibodies; anti-CD4antibodies; anti-CD52 antibodies; anti-Fc receptor antibodies;anti-carcinoembryonic antigen (CEA) antibodies; antibodies directedagainst breast epithelial cells; antibodies that bind to colon carcinomacells; anti-CD38 antibodies; anti-CD33 antibodies; anti-CD22 antibodies;anti-EpCAM antibodies; anti-GpIIb/IIIa antibodies; anti-RSV antibodies;anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies;anti-CA 125 antibodies; anti-αvβ3 antibodies; anti-human renal cellcarcinoma antibodies; anti-human 17-1A antibodies; anti-human colorectaltumor antibodies; anti-human melanoma antibody R24 directed against GD3ganglioside; anti-human squamous-cell carcinoma; and anti-humanleukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies.

In other embodiments, the therapeutic antibody is an antibody binding toa HER receptor, VEGF, IgE, CD20, CD11a, CD40, or DR5.

In a further embodiment, the HER receptor is HER1 and/or HER2,preferably HER2. The HER2 antibody may, for example, comprise a heavyand/or light chain variable domain sequence selected from the groupconsisting of SEQ ID NO: 16, 17, 18, and 19.

In another embodiment, the therapeutic antibody is an antibody thatbinds to CD20. The anti-CD20 antibody may, for example, comprise a heavyand/or light chain variable domain sequence selected from the groupconsisting of SEQ ID NOS: 1 through 15.

In yet another embodiment, the therapeutic antibody is an antibody thatbinds to VEGF.

The anti-VEGF antibody may, for example, comprise a heavy and/or lightchain variable domain sequence selected from the group consisting of SEQID NOS: 20 through 25.

In an additional embodiment, the therapeutic antibody is an antibodythat binds CD11a. The anti-CD11a antibody may, for example, comprise aheavy and/or light chain variable domain sequence selected from thegroup consisting of SEQ ID NOS: 26 through 29.

In a further embodiment, the therapeutic antibody binds to a DR5receptor. The anti-DR5 antibody may, for example, be selected from thegroup consisting of Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2,6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2,8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and 25.3, andpreferably is Apomab 8.3 or Apomab 7.3, and most preferably Apomab 7.3.

In other embodiments of the method of the present invention, thepolypeptide expressed in the recombinant host cell is a therapeuticpolypeptide. For example, the therapeutic polypeptide can be selectedfrom the group consisting of a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor, anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; ProteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-GF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor, viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of said polypeptides.

In all embodiments, the recombinant host cell can be an eukaryotic hostcell, such as a mammalian host cell, including, for example, ChineseHamster Ovary (CHO) cells.

In all embodiments, the recombinant host cell can also be a prokaryotichost cell, such as a bacterial cell, including, without limitation, E.coli cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and TrademarkOffice upon request and payment of the necessaryfees.

FIG. 1. Dialysis Experiment: Digital gel-like imaging obtained fromBioanalyzer analysis (each lane representing a time point) demonstratingthat ocrelizumab (rhuMAb 2H7—Variant A) inside the dialysis bag remainedintact during the incubation period.

FIG. 2. Dialysis Experiment: Digital gel-like imaging obtained fromBioanalyzer analysis (each lane representing a time point) showing thatocrelizumab outside the dialysis bag was reduced during the incubationperiod. This is evidenced by the loss of intact antibody (˜150 kDa) andthe formation of antibody fragments depicted in the Figure. At the48-hour time point (Lane 7), the reduced antibody appeared to bereoxidized, presumably as a result of loosing reduction activity in theHarvested Cell Culture Fluid (HCCF). The band appearing just above the28 kDa marker arose from the light chain of antibody. There was asignificant amount of free light already present in the HCCF before theincubation began. The presence of excess free light chain and dimers oflight chain in the HCCF is typical for the cell line producingocrelizumab.

FIG. 3. Free Thiol Levels from Dialysis Experiment: Purified ocrelizumabin phosphate buffered saline (PBS) was inside the dialysis bag and HCCFcontaining ocrelizumab was outside the bag. Free thiols inside (boxes)and outside (diamonds) the dialysis bag reached comparable levels withina few hours, indicating a good exchange of small molecule components inthe HCCF between inside and outside the dialysis bag.

FIG. 4. Thioredoxin System and Other Reactions Involved in AntibodyReduction: The thioredoxin system, comprising thioredoxin (Trx),thioredoxin reductase (TrxR) and NADPH, functions as a hydrogen donorsystem for reduction of disulfide bonds in proteins. Trx is a smallmonomeric protein with a CXXC active site motif that catalyzes manyredox reactions through thiol-disulfide exchange. The oxidized Trx canbe reduced by NADPH via TrxR. The reduced Trx is then able to catalyzethe reduction of disulfides in proteins. The NADPH required forthioredoxin system is provided via reactions in pentose phosphatepathway and glycolysis.

FIG. 5. In Vitro Activity of Thioredoxin System: Digital gel-like imagefrom Bioanalyzer analysis (each lane representing a time point)demonstrating that incubation of intact ocrelizumab (1 mg/mL) with 0.1mM TrxR (rat liver), 5 mM Trx (human), and 1 mM NADPH in PBS resulted inthe complete reduction of ocrelizumab; the ocrelizumab was completelyreduced in less than 21 hours.

FIG. 6. In Vitro Activity of Thioredoxin System Inhibited byAurothioglucose: The addition of aurothioglucose (ATG) to the samereaction mixture as described in the caption for FIG. 5, above,effectively inhibited the ocrelizumab reduction. This is seen by thedigital gel-like image from Bioanalyzer analysis (each lane representinga time point).

FIG. 7. In vitro Activity of Thioredoxin System Inhibited byAurothiomalate: The addition of aurothiomalate (ATM) at a concentrationof 1 mM to the same reaction mixture as described in the caption forFIG. 5, above, effectively inhibited the ocrelizumab reduction. This isseen by the digital gel-like image from Bioanalyzer analysis (each lanerepresenting a time point).

FIG. 8. In Vitro Activity of Thioredoxin System: Digital gel-like imagefrom Bioanalyzer analysis (each lane representing a time point) showingthat incubation of intact ocrelizumab (1 mg/mL) with 0.1 mM TrxR (ratliver), 5 mM Trx (human), and 1 mM NADPH in 10 mM histidine sulfatebuffer resulted in the reduction of ocrelizumab in less than 1 hour.

FIG. 9. In vitro Activity of Thioredoxin System Inhibited by CuSO₄: Theaddition of CuSO₄ at a concentration of 50 μM to the same reactionmixture as described in the caption for FIG. 8 effectively inhibited theocrelizumab reduction as shown in the digital gel-like image fromBioanalyzer analysis (each lane representing a time point).

FIG. 10. Ocrelizumab Reduction: Digital gel-like image from Bioanalyzeranalysis (each lane representing a time point) showing that ocrelizumabwas reduced in an incubation experiment using HCCF from a homogenizedCCF generated from a 3-L fermentor.

FIG. 11. Inhibition of Ocrelizumab Reduction In HCCF by Aurothioglucose:Digital gel-like image from Bioanalyzer analysis (each lane representinga time point) showing that the addition of 1 mM aurothioglucose to thesame HCCF as used for the incubation experiment as shown in FIG. 10inhibited the reduction of ocrelizumab.

FIG. 12. Inhibition of Ocrelizumab Reduction In HCCF by Aurothiomalate:Digital gel-like image from Bioanalyzer (each lane representing a timepoint) analysis indicating that the addition of 1 mM aurothiomalate tothe same HCCF as used for the incubation experiment shown in FIG. 10inhibited the reduction of ocrelizumab.

FIG. 13. Losing Reduction Activity in HCCF: The HCCF from one of thelarge scale manufacturing runs for ocrelizumab (the “beta” run) that wassubject to several freeze/thaw cycles demonstrated no ocrelizumabreduction when used in an incubation experiment. This was shown byBioanalyzer analysis (each lane representing a time point), and can becontrasted to the antibody reduction seen previously in the freshlythawed HCCF from the same fermentation batch.

FIG. 14. The Lost Reduction Activity in HCCF Restored by Addition ofNADPH: The reduction of ocrelizumab was observed again in theBioanalyzer assay (each lane representing a time point) after theaddition of NADPH at a concentration of 5 mM into the HCCF where thereduction activity has been eliminated under the conditions describedabove in FIG. 13.

FIG. 15. The Lost Reduction Activity in HCCF Restored by Addition ofGlucose-6-Phosphate: The reduction of ocrelizumab was observed again inthe Bioanalyzer assay (each lane representing a time point) after theaddition of G6P at a concentration of 10 mM into the HCCF where thereduction activity has been eliminated due to the treatment describedabove in FIG. 13.

FIG. 16. Ocrelizumab Reduction: A digital gel-like image fromBioanalyzer analysis showing that ocrelizumab was reduced in anincubation experiment using a HCCF from a large scale manufacturing run(the “alpha” run).

FIG. 17. EDTA Inhibits Ocrelizumab Reduction: Digital gel-like imagefrom Bioanalyzer analysis (each lane representing a time point) showingthat the reduction of ocrelizumab was inhibited in an incubationexperiment using a HCCF from the alpha run with EDTA added at aconcentration of 20 mM to the HCCF whose reducing activity isdemonstrated in FIG. 16.

FIG. 18. The Lost Reduction Activity in “Beta Run” HCCF Restored byAddition of Glucose-6-Phosphate but No Inhibition of Reduction by EDTA:The reduction of ocrelizumab was observed in the Bioanalyzer assay (eachlane representing a time point) after the addition of G6P at aconcentration of 5 mM and 20 mM EDTA into the HCCF whose reductionactivity had been lost (see FIG. 13). In contrast to the results shownin FIG. 17, the presence of EDTA did not block the reduction ofocreliumab.

FIG. 19. Inhibition of Ocrelizumab Reduction: by (i) addition of EDTA,(ii) addition of CuSO₄, or (iii) adjustment of pH to 5.5. All threedifferent methods, (1) addition of EDTA, (2) addition of CuSO₄, and (3)adjustment of pH to 5.5, used independently, were effective ininhibiting ocrelizumab reduction. This was demonstrated by the depictedquantitative Bioanalyzer results that showed that nearly 100% intact(150 kDa) antibody remained in the protein A elution pools. In contrast,ocrelizumab was completely reduced in the control HCCF after 20 hours ofHCCF hold time.

FIG. 20. Inhibition of Ocrelizumab Reduction by Air Sparging: Spargingthe HCCF with air was effective in inhibiting ocrelizumab disulfide bondreduction. This was demonstrated by the quantitative Bioanalyzer resultsshowing that nearly 100% intact (150 kDa) antibody remained in theprotein A elution pools. In contrast, ocrelizumab was almost completelyreduced in the control HCCF after 5 hours of sparging with nitrogen.

FIG. 21 shows the V_(L)(SEQ ID NO. 24) amino acid sequence of ananti-Her2 antibody (Trastuzumab).

FIG. 22 shows the V_(H) (SEQ ID No. 25) amino acid sequence of ananti-Her2 antibody (Trastuzumab).

FIG. 23 is a schematic showing some steps of a typical large scalemanufacturing process.

FIG. 24 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate.

FIG. 25 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 1 mM histidine sulfate+1 mM ATG.

FIG. 26 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+0.6 μM ATG (6:1 ATG:TrxR).

FIG. 27 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+0.4 μM ATG (4:1 ATG:TrxR).

FIG. 28 is a digital gel-like image from Bioanalyzer analysis: 2H7.(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+0.2 μM ATG (2:1 ATG:TrxR).

FIG. 29 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+0.1 mM autothiomalate (ATM).

FIG. 30 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+0.01 mM autothiomalate (ATM).

FIG. 31 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+20 μM CuSO₄ (4:1 Cu²⁺:Trx).

FIG. 32 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+10 μM CuSO₄ (2:1 Cu²⁺:Trx).

FIG. 33 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+5 μM CuSO₄ (1:1 Cu²⁺:Trx).

FIG. 34 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+532 μM cystamine (20:1cystamine:2H7 disulfide).

FIG. 35 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+266 μM cystamine (10:1cystamine:2H7 disulfide).

FIG. 36 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+133 μM cystamine (5:1cystamine:2H7 disulfide).

FIG. 37 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+26.6 μM cystamine (1:1cystamine:2H7 disulfide).

FIG. 38 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate (pH=7.6)+2.6 mM cystine.

FIG. 39 is a digital gel-like image from Bioanalyzer analysis: 2H7(Variant A)+1 mM NADPH+5 μM thioredoxin+0.1 μM thioredoxin reductase(recombinant) in 10 mM histidine sulfate+2.6 mM GSSG (oxidizedglutathione).

FIG. 40 Reconstructed enzymatic reduction system. 1 mg/ml 2H7 (VariantA)+10 μg/mL hexokinase, 50 μg/mL glucose-6-phosphate dehydrogenase, 5 μMthioredoxin, 0.1 μM thioredoxin reductase, 2 mM glucose, 0.6 mM ATP, 2mM Mg²⁺, and 2 mM NADP in 50 mM histidine sulfate buffer at pH=7.38.

FIG. 41 The thioredoxin system requires NADPH. 1 mg/ml 2H7 (Variant A)+5μM thioredoxin, 0.1 μM thioredoxin reductase, and 2 mM NADP in 50 mMhistidine sulfate buffer at pH=7.38.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

In the present invention, in the context of proteins, includingantibodies, in general, or with regard to any specific protein orantibody, the term “reduction” is used to refer to the reduction of oneor more disulfide bonds of the protein or antibody. Thus, for example,the terms “ocrelizumab reduction” is used interchangeably with the term“ocrelizumab disulfide bond reduction” and the term “antibody (Ab)reduction” is used interchangeably with the term “antibody (Ab)disulfide bond reuction.”

The terms “reduction” or “disulfide bond reduction” are used in thebroadest sense, and include complete and partial reduction and reductionof some or all of the disulfide bonds, interchain or intrachain, presentin a protein such as an antibody.

By “protein” is meant a sequence of amino acids for which the chainlength is sufficient to produce the higher levels of tertiary and/orquaternary structure. This is to distinguish from “peptides” or othersmall molecular weight drugs that do not have such structure. Typically,the protein herein will have a molecular weight of at least about 15-20kD, preferably at least about 20 kD. Examples of proteins encompassedwithin the definition herein include all mammalian proteins, inparticular, therapeutic and diagnostic proteins, such as therapeutic anddiagnostic antibodies, and, in general proteins that contain one or moredisulfide bonds, including multi-chain polypeptides comprising one ormore inter- and/or intrachain disulfide bonds.

The term “therapeutic protein” or “therapeutic polypeptide” refers to aprotein that is used in the treatment of disease, regardless of itsindication or mechanism of action. In order for therapeutic proteins tobe useful in the clinic it must be manufactured in large quantities.“Manufacturing scale” production of therapeutic proteins, or otherproteins, utilize cell cultures ranging from about 400 L to about 80,000L, depending on the protein being produced and the need. Typically suchmanufacturing scale production utilizes cell culture sizes from about400 L to about 25,000 L. Within this range, specific cell culture sizessuch as 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000L, about 14,000 L, or about 16,000 L are utilized.

The term “therapeutic antibody” refers to an antibody that is used inthe treatment of disease.

A therapeutic antibody may have various mechanisms of action. Atherapeutic antibody may bind and neutralize the normal function of atarget associated with an antigen. For example, a monoclonal antibodythat blocks the activity of the of protein needed for the survival of acancer cell causes the cell's death. Another therapeutic monoclonalantibody may bind and activate the normal function of a targetassociated with an antigen. For example, a monoclonal antibody can bindto a protein on a cell and trigger an apoptosis signal. Yet anothermonoclonal antibody may bind to a target antigen expressed only ondiseased tissue; conjugation of a toxic payload (effective agent), suchas a chemotherapeutic or radioactive agent, to the monoclonal antibodycan create an agent for specific delivery of the toxic payload to thediseased tissue, reducing harm to healthy tissue. A “biologicallyfunctional fragment” of a therapeutic antibody will exhibit at least oneif not some or all of the biological functions attributed to the intactantibody, the function comprising at least specific binding to thetarget antigen.

The term “diagnostic protein” refers to a protein that is used in thediagnosis of a disease.

The term “diagnostic antibody” refers to an antibody that is used as adiagnostic reagent for a disease. The diagnostic antibody may bind to atarget antigen that is specifically associated with, or shows increasedexpression in, a particular disease. The diagnostic antibody may beused, for example, to detect a target in a biological sample from apatient, or in diagnostic imaging of disease sites, such as tumors, in apatient. A “biologically functional fragment” of a diagnostic antibodywill exhibit at least one if not some or all of the biological functionsattributed to the intact antibody, the function comprising at leastspecific binding to the target antigen.

“Purified” means that a molecule is present in a sample at aconcentration of at least 80-90% by weight of the sample in which it iscontained.

The protein, including antibodies, which is purified is preferablyessentially pure and desirably essentially homogeneous (i.e. free fromcontaminating proteins etc.).

An “essentially pure” protein means a protein composition comprising atleast about 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight.

An “essentially homogeneous” protein means a protein compositioncomprising at least about 99% by weight of protein, based on totalweight of the composition.

As noted above, in certain embodiments, the protein is an antibody.“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length antibodies whichhave an immunoglobulin Fc region), antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules such as scFv molecules, as well as antibodyfragments (e.g., Fab, F(ab′)₂, and Fv).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies.i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (see,e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J.Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132(2004), andtechnologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893;WO9634096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Nat. Acad.Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Markset al., BioTechnology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all theFRs are those of a human immunoglobulin sequence. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992). See also the following review articles and referencescited therein: Vaswani and Hamilton, Ann. Allergy. Asthma & Immunol.1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038(1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). Thehumanized antibody includes a Primatized™ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs/HVRs thereof which result in an improvement in theaffinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by V_(H) and V_(L) domain shuffling.Random mutagenesis of CDR/HVR and/or framework residues is described by:Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier etal. Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992).

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in the binding of an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called a, d, e, g, and m, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise an antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, the scFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Pluckthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO93/1161; Hudson et al., (2003) Nat. Med. 9:129-134; andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., (2003)Nat. Med. 9:129-134.

The antibody may bind to any protein, including, without limitation, amember of the HER receptor family, such as HER1 (EGFR), HER2, HER3 andHER4; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD21, CD22, andCD34; cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM and av/p3 integrin including either α or β or subunitsthereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); growthfactors such as vascular endothelial growth factor (VEGF); IgE; bloodgroup antigens; flk2/flt3 receptor, obesity (OB) receptor; and proteinC. Other exemplary proteins include growth hormone (GH), including humangrowth hormone (hGH) and bovine growth hormone (bGH); growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; α-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor, tissuefactor, and von Willebrands factor, anti-clotting factors such asProtein C; atrial natriuretic factor, lung surfactant; a plasminogenactivator, such as urokinase or tissue-type plasminogen activator(t-PA); bombazine; thrombin; tumor necrosis factor-α and -β;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-α); serumalbumin such as human serum albumin (HSA); mullerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; DNase; inhibin; activin; receptors forhormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-α and TGF-β,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I);insulin-like growth factor binding proteins (IGFBPs); erythropoietin(EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; abone morphogenetic protein (BMP); an interferon such as interferon-α,-β, and -γ, colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, andG-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;T-cell receptors; surface membrane proteins; decay accelerating factor(DAF); a viral antigen such as, for example, a portion of the AIDSenvelope; transport proteins; homing receptors; addressins; regulatoryproteins; immunoadhesins; antibodies; and biologically active fragmentsor variants of any of the above-listed polypeptides. Many otherantibodies and/or other proteins may be used in accordance with theinstant invention, and the above lists are not meant to be limiting.

A “biologically functional fragment” of an antibody comprises only aportion of an intact antibody, wherein the portion retains at least one,and as many as most or all, of the functions normally associated withthat portion when present in an intact antibody. In one embodiment, abiologically functional fragment of an antibody comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, a biologically functional fragment of anantibody, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibody halflife modulation, ADCC function and complement binding. In oneembodiment, a biologically functional fragment of an antibody is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For example, such a biologically functionalfragment of an antibody may comprise an antigen binding arm linked to anFc sequence capable of conferring in vivo stability to the fragment.

The terms “thioredoxin inhibitor” and “Trx inhibitor” are usedinterchangeably, and include all agents and measures effective ininhibiting thioredoxin activity. Thus, thioredoxin (Trx) inhibitorsinclude all agents and measures blocking any component of the Trx, G6PDand/or hexokinase enzyme systems. In this context, “inhibition” includescomplete elimination (blocking) and reduction of thioredoxin activity,and, consequently, complete or partial elimination of disulfide bondreduction in a protein, such as an antibody.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a C_(H)2/C_(H)3 region, such as an Fc region.Protein A can be purchased commercially from Repligen, GE Healthcare andFermatech. Protein A is generally immobilized on a solid phase supportmaterial. The term “ProA” also refers to an affinity chromatographyresin or column containing chromatographic solid support matrix to whichis covalently attached Protein A.

The term “chromatography” refers to the process by which a solute ofinterest in a mixture is separated from other solutes in a mixture as aresult of differences in rates at which the individual solutes of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably herein and refer to a protein separationtechnique in which a protein of interest or antibody of interest isreversibly and specifically bound to a biospecific ligand. Preferably,the biospecific ligand is covalently attached to a chromatographic solidphase material and is accessible to the protein of interest in solutionas the solution contacts the chromatographic solid phase material. Theprotein of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein of interest is then removed inactive form from the immobilized ligand with low pH, high pH, high salt,competing ligand, and the like, and passed through the chromatographiccolumn with the elution buffer, free of the contaminating proteins orprotein impurities that were earlier allowed to pass through the column.Any component can be used as a ligand for purifying its respectivespecific binding protein, e.g. antibody.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification process in which affinity chromatography is notutilized. Non-affinity chromatography includes chromatographictechniques that rely on non-specific interactions between a molecule ofinterest (such as a protein, e.g. antibody) and a solid phase matrix.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include carboxy-methyl-cellulose, sulphopropyl(SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW™ orSP-SEPHAROSE HIGH PERFORMANCE™, from GE Healthcare) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW™ from GE Healthcare).A “mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties. Acommercially available mixed mode ion exchange resin is BAKERBOND ABX™(J. T. Baker, Phillipsburg, N.J.) containing weak cation exchangegroups, a low concentration of anion exchange groups, and hydrophobicligands attached to a silica gel solid phase support matrix.

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX™and FAST Q SEPHAROSE™ (GE Healthcare).

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In oneembodiment, the buffer has a pH in the range from about 2 to about 9,alternatively from about 3 to about 8, alternatively from about 4 toabout 7 alternatively from about 5 to about 7. Non-limiting examples ofbuffers that will control the pH in this range include MES, MOPS, MOPSO,Tris, HEPES, phosphate, acetate, citrate, succinate, and ammoniumbuffers, as well as combinations of these.

The “loading buffer” is that which is used to load the compositioncomprising the polypeptide molecule of interest and one or moreimpurities onto the ion exchange resin. The loading buffer has aconductivity and/or pH such that the polypeptide molecule of interest(and generally one or more impurities) is/are bound to the ion exchangeresin or such that the protein of interest flows through the columnwhile the impurities bind to the resin.

The “intermediate buffer” is used to elute one or more impurities fromthe ion exchange resin, prior to eluting the polypeptide molecule ofinterest. The conductivity and/or pH of the intermediate buffer is/aresuch that one or more impurity is eluted from the ion exchange resin,but not significant amounts of the polypeptide of interest.

The term “wash buffer” when used herein refers to a buffer used to washor re-equilibrate the ion exchange resin, prior to eluting thepolypeptide molecule of interest. Conveniently, the wash buffer andloading buffer may be the same, but this is not required.

The “elution buffer” is used to elute the polypeptide of interest fromthe solid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all impurities and thepolypeptide of interest from the ion exchange resin.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein with regard to amounts or numerical values (and not asreference to the chemical process of reduction), denotes a sufficientlyhigh degree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is, for example, greater than about 10%, greater than about20%, greater than about 30%, greater than about 40%, and/or greater thanabout 50% as a function of the value for the reference/comparatormolecule.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

-   -   100 times the fraction X/Y    -   where X is the number of amino acid residues scored as identical        matches by the sequence alignment program ALIGN-2 in that        program's alignment of A and B, and    -   where Y is the total number of amino acid residues in B.        It will be appreciated that where the length of amino acid        sequence A is not equal to the length of amino acid sequence B,        the % amino acid sequence identity of A to B will not equal the        % amino acid sequence identity of B to A. Unless specifically        stated otherwise, all % amino acid sequence identity values used        herein are obtained as described in the immediately preceding        paragraph using the ALIGN-2 computer program.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference Factor D-encoding sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST. BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented. “Treatment” herein encompasses alleviation of the disease andof the signs and symptoms of the particular disease.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. Non-limiting examples of disorders to be treatedherein include carcinomas and allergies.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, non-human higher primates, other vertebrates,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, horses, cats, cows, etc. Preferably, the mammal is human.

An “interfering RNA” or “small interfering RNA (siRNA)” is a doublestranded RNA molecule less than about 30 nucleotides in length thatreduces expression of a target gene. Interfering RNAs may be identifiedand synthesized using known methods (Shi Y., Trends in Genetics19(1):9-12 (2003), WO/2003056012 and WO2003064621), and siRNA librariesare commercially available, for example from Dharmacon, Lafayette, Colo.Frequently, siRNAs can be successfully designed to target the 5′ end ofa gene.

II. Compositions and Methods of the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology and the like,which are within the skill of the art. Such techniques are explainedfully in the literature. See e.g., Molecular Cloning: A LaboratoryManual, (J. Sambrook et al., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubelet al., eds., 1987 updated); Essential Molecular Biology (T. Brown ed.,IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwellet al., eds., Bartlett Publ. 1990); Gene Transfer and Expression (M.Kriegler, Stockton Press 1990); Recombinant DNA Methodology II (R. Wu etal., eds., Academic Press 1995); PCR: A Practical Approach (M. McPhersonet al., IRL Press at Oxford University Press 1991); OligonucleotideSynthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adamsed., Elsevier Science Publishers 1990); Gene Transfer Vectors forMammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian CellBiotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard etal., eds., Humana Press 1990); Culture of Animal Cells, 2^(nd) Ed. (R.Freshney et al., eds., Alan R. Liss 1987); Flow Cytometry and Sorting(M. Melamed et al., eds., Wiley-Liss 1990); the series Methods inEnzymology (Academic Press, Inc.); Wirth M. and Hauser H. (1993);Immunochemistry in Practice, 3rd edition, A. Johnstone & R. Thorpe,Blackwell Science, Cambridge, Mass., 1996; Techniques inImmunocytochemistry, (G. Bullock & P. Petrusz eds., Academic Press 1982,1983, 1985, 1989); Handbook of Experimental Immunology, (D. Weir & C.Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al.,eds. 1991); Immunoassay (E. P. Diamandis & T. K. Christopoulos, eds.,Academic Press, Inc., 1996); Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed) Academic Press, New York; Ed Harlow andDavid Lane, Antibodies A laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988; Antibody Engineering, 2^(nd)edition (C. Borrebaeck, ed., Oxford University Press, 1995); and theseries Annual Review of Immunology; the series Advances in Immunology.

1. Prevention of Disulfide Bond Reduction

The present invention concerns methods for the prevention of thereduction of disulfide bonds of proteins during recombinant production.In particular, the invention concerns methods for preventing thereduction of disulfide bonds of recombinant proteins during processingfollowing fermentation. The methods of the invention are particularlyvaluable for large scale production of disulfide bond containingproteins, such as at a manufacturing scale. In one embodiment, themethods of the invention are useful for large scale protein productionat a scale of greater than 5,000 L.

It has been experimentally found that disulfide bond reduction occursduring processing of the Harvested Cell Culture Fluid (HCCF) producedduring manufacturing of recombinant proteins that contain disulfidebonds. Typically, this reduction is observed after cell lysis,especially mechanical cell lysis during harvest operations, when itreaches a certain threshold, such as, for example, from about 30% toabout 70%, or from about 40% to about 60%, or from about 50% to about60% total cell lysis. This threshold will vary, depending on the natureof the protein (e.g. antibody) produced, the recombinant host, theproduction system, production parameters used, and the like, and can bereadily determined experimentally.

Theoretically, such reduction might result from a variety of factors andconditions during the manufacturing process, and might be caused by avariety of reducing agents. The present invention is based, at least inpart, on the recognition that the root cause of this reduction is anactive thioredoxin (Trx) or thioredoxin-like system in the HCCF.

The Trx enzyme system, composed of Trx, thioredoxin reductase (TrxR) andNADPH, is a hydrogen donor system for reduction of disulfide bonds inproteins. Trx is a small monomeric protein with a CXXC active site motifthat catalyzes many redox reactions through thiol-disulfide exchange.The oxidized Trx can be reduced by NADPH via TrxR. The reduced Trx isthen able to catalyze the reduction of disulfides in proteins. The NADPHrequired for thioredoxin system is provided via reactions in pentosephosphate pathway and glycolysis. The results presented hereindemonstrate that NADPH, which is required for activity of the Trx systemis provided by glucose-6-phosphate dehyrogenase (G6PD) activity, whichgenerates NADPH from glucose and ATP by hexokinase (see FIG. 4). Thesecellular enzymes (Trx system, G6PD, and hexokinase) along with theirsubstrates are released into the CCF upon cell lysis, allowing reductionto occur. Accordingly, disulfide reduction can be prevented byinhibitors of the Trx enzyme system or upstream enzyme systems providingcomponents for an active Trx system, such as G6PD and hexokinaseactivity.

For further details of these enzyme systems, or regarding other detailsof protein production, see, for example: Babson, A. L. and Babson, S. R.(1973) Kinetic Colorimetric Measurement of Serum Lactate DehydrogenaseActivity. Clin. Chem. 19: 766-769; Michael W. Laird et al.,“Optimization of BLyS Production and Purification from Eschericia coli,”Protein Expression and Purification 39:237-246 (2005); John C. Joly etal., “Overexpression of Eschericia coli Oxidoreductases IncreasesRecombinant Insulin-like Growth Factor-I Accumulation,” Proc. Natl.Acad. Sci. USA 95:2773-2777 (March 1998); Dana C. Andersen et al.,“Production Technologies for Monoclonal Antibodies and Their Fragments,”Current Opinion in Biotechnology 15:456-462 (2004); Yariv Mazor et al.,“Isolation of Engineered, Full-length Antibodies from LibrariesExpressed in Escherichia coli,” Nature Biotech. 25, 563-565 (1 Jun.2007); Laura C. Simmons et al., “Expression of Full-lengthImmunoglobulins in Escherichia coli: Rapid and Efficient Production ofAglycosylated Antibodies,” Journal of Immunological Methods 263:133-147(2002); Paul H. Bessette et al., “Efficient Folding of Proteins withMultiple Disulfide Bonds in the Escherichia coli cytoplasm,” Proc. Nat.Acad. Sci. 96(24):13703-08 (1999); Chaderjian, W. B., Chin, E. T.,Harris, R. J., and Etcheverry, T. M., (2005) “Effect of copper sulfateon performance of a serum-free CHO cell culture process and the level offree thiol in the recombinant antibody expressed,” Biotechnol. Pro. 21:550-553; Gordon G., Mackow M. C., and Levy H. R., (1995) “On themechanism of interaction of steroids with human glucose 6-phosphatedehydrogenase,” Arch. Biochem. Biophys. 318: 25-29; Gromer S., Urig S.,and Becker K., (2004) “TheTrx System—From Science to Clinic,” MedicinalResearch Reviews, 24: 40-89; Hammes G. G. and Kochavi D., (1962a)“Studies of the Enzyme Hexokinase. I. Steady State Kinetics at pH 8,” J.Am. Chem. Soc. 84:2069-2073; Hammes G. G. and Kochavi D., (1962b)“Studies of the Enzyme Hexokinase. III. The Role of the Metal Ion,” J.Am. Chem. Soc. 84:2076-2079; Johansson C., Lillig C. H., and HolmgrenA., (2004) “Human Mitochondrial Glutaredoxin Reduces S-GlutathionylatedProteins with High Affinity Accepting Electrons from Either Glutathioneor Thioredoxin Reductase,” J. Biol. Chem. 279:7537-7543; Legrand, C.,Bour, J. M., Jacob, C., Capiaumont J., Martial, A., Marc, A., Wudtke,M., Kretzmer, G., Demangel, C., Duval, D., and Hache J., (1992) “LactateDehydrogenase (LDH) Activity of the Number of Dead Cells in the Mediumof Cultured Eukaryotic Cells as Marker,” J. Biotechnol., 25: 231-243;McDonald, M. R., (1955) “Yeast Hexokinase:ATP+Hexose-->Hexose-6-phosphate+ADP,” Methods in Enzymology, 1: 269-276,Academic Press, NY; Sols, A., DelaFuente, G., Villar-Palasi, C., andAsensio, C., (1958) “Substrate Specificity and Some Other Properties ofBakers' Yeast Hexokinase,” Biochim Biophys Acta 30: 92-101; KirkpatrickD. L., Kupeus M., Dowdeswell M., Potier N., Donald L. J., Kunkel M.,Berggren M., Angulo M., and Powis G., (1998) “Mechanisms of inhibitionof the Trx growth factor system by antitumor 2-imidazolyl disulfides,”Biochem. Pharmacol. 55: 987-994; Kirkpatrick D. L., Watson S., KunkelM., Fletcher S., Ulhaq S., and Powis G., (1999) “Parallel syntheses ofdisulfide inhibitors of the Trx redox system as potential antitumoragents,” Anticancer Drug Des. 14: 421-432; Milhausen, M., and Levy, H.R., (1975) “Evidence for an Essential Lysine in G6PD from Leuconostocmesenteroides,” Eur. J. Biochem. 50: 453-461; Pleasants, J. C., Guo, W.,and Rabenstein, D. L., (1989) “A comparative study of the kinetics ofselenol/diselenide and thiol/disulfide exchange reactions,” J. Am. Chem.Soc. 111: 6553-6558; Whitesides, G. M., Lilbum, J. E., and Szajewski, R.P., (1977) “Rates of thioldisulfide interchange reactions between mono-and dithiols and Eliman's reagent,” J. Org. Chem. 42: 332-338; and WipfP., Hopkins T. D., Jung J. K., Rodriguez S., Birmingham A., Southwick E.C., Lazo J. S., and Powis G, (2001) “New inhibitors of the Trx-TrxRsystem based on a naphthoquinone spiroketal natural product lead,”Bioorg. Med. Chem. Lett. 11: 2637-2641.

According to one aspect of the present invention, disulfide bondreduction can be prevented by blocking any component of the Trx, G6PDand hexokinase enzyme systems. Inhibitors of these enzyme systems arecollectively referred to herein as “thioredoxin inhibitors,” or “Trxinhibitors.” The Trx inhibitors are typically added to the cell culturefluid (CCF), which contains the recombinant host cells and the culturemedia, and/or to the harvested cell culture fluid (HCCF), which isobtained after harvesting by centrifugation, filtration, or similarseparation methods. The HCCF lacks intact host cells but typicallycontains host cell proteins and other contaminants, including DNA, whichare removed in subsequent purification steps. Thus, the Trx inhibitorsmay be added before harvest and/or during harvest, preferably beforeharvest.

Alternatively or in addition other, non-specific methods can also beused to prevent the reduction of disulfide bond reduction followingfermentation during the recombinant production of recombinant proteins,such as air sparging or pH adjustment. Certain reduction inhibitionmethods contemplated herein are listed in the following Table 1.

TABLE 1 Reduction Inhibition Methods Method¹ Purpose Addition of EDTA,EGTA, or To inhibit hexokinase citrate Addition of sorbose-1-phosphate,To inhibit hexokinase polyphosphates, 6-deoxy-6- fluoroglucose,2-C-hydroxy- methylglucose, xylose, or lyxose Addition ofepiandrosterone or To inhibit G6PD dehydroepiandrosterone (DHEA)Addition of pyridoxal 5′-phosphate To inhibit G6PD or1-fluoro-2,4-dinitrobenzene Addition of metal ions such as To inhibitTrx system Cu²⁺, Zn²⁺ Hg²⁺, Co²⁺, or Mn²⁺ Addition of alkyl-2-imidazolylTo inhibit Trx disulfides and related compounds (e.g., 1methylpropyl-2-imidazolyl disulfide²) or naphthoquinone spiroketalderivatives (e.g. palmarumycin CP₁ ²⁾ Addition of aurothioglucose (ATG)To inhibit TrxR or aurothiomalate (ATM) Air sparging To deplete G6P andNADPH; oxidizing agent Cystine Oxidizing agent Oxidized glutathioneOxidizing agents pH Adjustment to below 6.0 To reduce thiol-disulfideexchange rate and Trx system activity ¹Applied to CCF prior to harvestor in HCCF immediately after harvest. ²Currently not availablecommercially.

“Trx inhibitors” for use in the methods of the present inventioninclude, without limitation, (1) direct inhibitors of Trx, such asalkyl-2-imidazolyl disulfides and related compounds (e.g., 1methylpropyl-2-imidazolyl disulfide) (Kirkpatrick et al., 1998 and 1999,supra) and naphthoquinone spiroketal derivatives (e.g., palmarumycinCP₁) (Wipf et al., 2001, supra); (2) specific inhibitors of TrxR,including gold complexes, such as aurothioglucose (ATG) andaurothiomalate (ATM) (see, e.g., the review by Gromer et al., 2004),which are examples of irreversible inhibitors of TrxR; (3) metal ions,such as Hg²⁺, Cu²⁺, Zn²⁺, Co²⁺, and Mn²⁺, which can form readilycomplexes with thiols and selenols, and thus can be used in embodimentsof the instant invention as inhibitors of TrxR or Trx; (4) inhibitors ofG6PD, such as, for example, pyridoxal 5′-phosphate and 1 fluoro-2,4dinitrobenzene (Milhausen and Levy 1975, supra), certain steroids, suchas dehydroepiandrosterone (DHEA) and epiandrosterone (EA) (Gordon etal., 1995, supra); and (4) inhibitors of hexokinase activity (andthereby production of G6P for the G6PD), including chelators of metalions, e.g. Mg²⁺, such as EDTA, and compounds that react with SH groups,sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose,2-C-hydroxy-methylglucose, xylose and lyxose (Sols et al., 1958, supra;McDonald, 1955, supra); further hexokinase inhibitors are disclosed inU.S. Pat. No. 5,854,067 entitled “Hexokinase Inhibitors.” It will beunderstood that these inhibitors are listed for illustration only. OtherTrx inhibitors exists and can be used, alone or in various combinations,in the methods of the present invention.

“Trx inhibitors” for use in the methods of the present invention alsoinclude reagents whereby the reduction of recombinantly producedantibodies or proteins may be reduced or prevented by decreasing thelevels of enzymes of the Trx system, the pentose phosphate pathway orhexokinase at various points during the production campaign. In someembodiments, this reduction of enzyme levels may be accomplished by theuse of targeted siRNAs, antisense nucleotides, or antibodies. To designtargeted siRNAs or antisense nucleotides to the genes as found in CHOcells, these gene sequences are available from public databases toselect sequences for targeting enzymes in different organisms. SeeExample 9 below for examples of the genes of the E. coli and mouse Trxsystem.

In addition to using inhibitors discussed above, it is also possible incertain embodiments of the instant invention to prevent the reduction ofa recombinant protein to be purified by sparging the HCCF with air tomaintain an oxidizing redox potential in the HCCF. This is anon-directed measure that can deplete glucose, G6P and NADPH bycontinuously oxidizing the reduced forms of Trx and TrxR. Air spargingof the HCCF tank can be performed, for example, with an air flow ofabout 100 liters to about 200 liters, such as, for example, 150 litersper minutes. Air sparging can be performed to reach an endpointpercentage of saturation; for example, air sparging can be continueduntil the HCCF is about 100% saturated with air, or it can be continueduntil the HCCR is about 30% saturated with air, or until it is betweenabout 100% saturated to about 30% saturated with air. The minimum amountof dissolved oxygen (dO₂) required for the desired inhibitory effectalso depends on the antibody or other recombinant protein produced.Thus, for example, about 10% dO₂ (or about 10 sccm for continuousstream) will have the desired effect during the production of antibody2H7 (Variant A), while Apomab might require a higher (about 30%) dO₂.

In further embodiments of the instant invention, another non-directedmethod usable to block the reduction of the recombinant protein islowering the pH of the HCCF. This embodiment takes advantage ofparticularly slow thiol-disulfide exchange at lower pH values(Whitesides et al., 1977, supra; Pleasants et al., 1989, supra).Therefore, the activity of the Trx system is significantly lower at pHvalues below 6, and thus the reduction of the recombinant protein, suchas ocrelizumab, can be inhibited.

The non-directed approaches can also be combined with each other and/orwith the use of one or more Trx inhibitors.

Disulfide bond reduction can be inhibited (i.e., partially or fullyblocked) by using one or more Trx inhibitors and/or applyingnon-directed approaches following completion of the cell cultureprocess, preferably to CCF prior to harvest or in the HCCF immediatelyafter harvest. The optimal time and mode of application and effectiveamounts depend on the nature of the protein to be purified, therecombinant host cells, and the specific production method used.Determination of the optimal parameters is well within the skill ofthose of ordinary skill in the art.

For example, in a mammalian cell culture process, such as the CHOantibody production process described in the Examples herein, if cupricsulfate (CuSO₄ in the form of pentahydrate or the anhydrous form) isused as a Trx inhibitor, it can be added to supplement the CCF or HCCFin the concentration range of from about 5 μM to about 100 μM, such asfrom about 10 μM to about 80 μM, preferably from about 15 μM to about 50μM. Since some cell cultures already contain copper (e.g. about 0.04 μMCuSO₄ for the CHO cell cultures used in the Examples herein), thisamount is in addition to the copper, if any, already present in the cellculture. Any copper (II) salt can be used instead of CuSO₄ as long assolubility is not an issue. For example, copper acetate and copperchloride, which are both soluble in water, can be used instead of CuSO₄.The minimum effective concentration may also depend on the antibodyproduced and the stage where the inhibitor is used. Thus, for example,when cupric sulfate is added pre-lysis, for antibody 2H7 (Variant A) theminimum effective concentration is about 30 μM, for Apomab is about 75JIM, and for antibody Variant C (see Table 2) is about 50 μM. Whencupric sulfate is added in CC medium, for antibody 2H7 (Variant A) theminimum effective concentration is about 15 μM, for Apomab is about 25μM, and for antibody Variant C is about 20 μM. One typical minimal CuSO₄inhibitor concentration of 2×Trx concentration (or Trx equivalence).

EDTA can be used in a wide concentration range, depending on the extentof cell lysis, the recombinant host cell used, and other parameters ofthe production process. For example, when using CHO or other mammalianhost cells, EDTA can be typically added in a concentration of betweenabout 5 mM to about 60 mM, such as from about 10 mM to about 50 mM, orfrom about 20 mM to about 40 mM, depending on the extent of cell lysis.For lower degree of cell lysis, lower concentrations of EDTA willsuffice, while for a cell lysis of about 75%-100%, the required EDTAconcentration is higher, such as, for example, from about 20 mM to about40 mM. The minimum effective concentration may also depend on theantibody produced. Thus, for example, for antibody 2H7 (Variant A) theminimum effective EDTA concentration is about 10 mM.

DHEA as a Trx inhibitor is typically effective at a lower concentration,such as for example, in the concentration range from about 0.05 mM toabout 5 mM, preferably from about 0.1 mM to about 2.5 mM.

Other Trx inhibitors, such as aurothioglucose (ATG) and aurothiomalate(ATM) inhibit reduction of disulfide bonds in the μM concentrationrange. Thus, for example, ATG or ATM may be added in a concentrationbetween about 0.1 mM to about 1 mM. While the minimum inhibitoryconcentration varies depending on the actual conditions, for ATG and ATMtypically it is around 4×TrxR concentration.

It is noted that all inhibitors can be used in an excess amount,therefore, it is not always necessary to know the amount of Trx or TrxRin the system.

In a preferred embodiment, the mammalian host cell used in themanufacturing process is a chinese hamster ovary (CHO) cell (Urlaub etal., Proc. Natl. Acad Sci. USA 77:4216 (1980)). Other mammalian hostcells include, without limitation, monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture), Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);mouse sertoli cells (TM4, Mather, Biol. Reprod 23:243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982)); MRC 5 cells; FS4 cells; a human hepatoma line (Hep G2); andmyeloma or lymphoma cells (e.g. Y0, J558 L, P3 and NS0 cells)(see U.S.Pat. No. 5,807,715).

A preferred host cell for the production of the polypeptides herein isthe CHO cell line DP12 (CHO K1 dhfr-). This is one of the best known CHOcell lines, widely used in laboratory practice (see, for example, EP0,307,247, published Mar. 15, 1989). In addition, other CHO-K1 (dhfr)cell lines are known and can be used in the methods of the presentinvention.

The mammalian host cells used to produce peptides, polypeptides andproteins can be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace (1979), Meth. in Enz.58:44, Barnes and Sato (1980), Anal. Biochem. 102:255, U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195;U.S. Pat. No. Re. 30,985; or U.S. Pat. No. 5,122,469, the disclosures ofall of which are incorporated herein by reference, may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

A protocol for the production, recovery and purification of recombinantantibodies in mammalian, such as CHO, cells may include the followingsteps: Cells may be cultured in a stirred tank bioreactor system and afed batch culture, procedure is employed. In a preferred fed batchculture the mammalian host cells and culture medium are supplied to aculturing vessel initially and additional culture nutrients are fed,continuously or in discrete increments, to the culture during culturing,with or without periodic cell and/or product harvest before terminationof culture. The fed batch culture can include, for example, asemi-continuous fed batch culture, wherein periodically whole culture(including cells and medium) is removed and replaced by fresh medium.Fed batch culture is distinguished from simple batch culture in whichall components for cell culturing (including the cells and all culturenutrients) are supplied to the culturing vessel at the start of theculturing process. Fed batch culture can be further distinguished fromperfusion culturing insofar as the supernate is not removed from theculturing vessel during the process (in perfusion culturing, the cellsare restrained in the culture by, e.g., filtration, encapsulation,anchoring to microcarriers etc. and the culture medium is continuouslyor intermittently introduced and removed from the culturing vessel).

Further, the cells of the culture may be propagated according to anyscheme or routine that may be suitable for the particular host cell andthe particular production plan contemplated. Therefore, a single step ormultiple step culture procedure may be employed. In a single stepculture the host cells are inoculated into a culture environment and theprocesses are employed during a single production phase of the cellculture. Alternatively, a multi-stage culture can be used. In themulti-stage culture cells may be cultivated in a number of steps orphases. For instance, cells may be grown in a first step or growth phaseculture wherein cells, possibly removed from storage, are inoculatedinto a medium suitable for promoting growth and high viability. Thecells may be maintained in the growth phase for a suitable period oftime by the addition of fresh medium to the host cell culture.

In certain embodiments, fed batch or continuous cell culture conditionsmay be devised to enhance growth of the mammalian cells in the growthphase of the cell culture. In the growth phase cells are grown underconditions and for a period of time that is maximized for growth.Culture conditions, such as temperature, pH, dissolved oxygen (dO₂) andthe like, are those used with the particular host and will be apparentto the ordinarily skilled artisan. Generally, the pH is adjusted to alevel between about 6.5 and 7.5 using either an acid (e.g., CO₂) or abase (e.g., Na2CO₃ or NaOH). A suitable temperature range for culturingmammalian cells such as CHO cells is between about 30° C. to 38° C., anda suitable dO₂ is between 5-90% of air saturation.

At a particular stage the cells may be used to inoculate a productionphase or step of the cell culture. Alternatively, as described above theproduction phase or step may be continuous with the inoculation orgrowth phase or step.

The cell culture environment during the production phase of the cellculture is typically controlled. Thus, if a glycoprotein is produced,factors affecting cell specific productivity of the mammalian host cellmay be manipulated such that the desired sialic acid content is achievedin the resulting glycoprotein. In a preferred aspect, the productionphase of the cell culture process is preceded by a transition phase ofthe cell culture in which parameters for the production phase of thecell culture are engaged. Further details of this process are found inU.S. Pat. No. 5,721,121, and Chaderjian et al., Biotechnol. Prog.21(2):550-3 (2005), the entire disclosures of which are expresslyincorporated by reference herein.

Following fermentation proteins are purified. Procedures forpurification of proteins from cell debris initially depend on the siteof expression of the protein. Some proteins can be caused to be secreteddirectly from the cell into the surrounding growth media; others aremade intracellularly. For the latter proteins, the first step of apurification process involves lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.The same problem arises, although on a smaller scale, with directlysecreted proteins due to the natural death of cells and release ofintracellular host cell proteins and components in the course of theprotein production run.

Once a clarified solution containing the protein of interest has beenobtained, its separation from the other proteins produced by the cell isusually attempted using a combination of different chromatographytechniques. These techniques separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down alongcolumn, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different solvents. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through.” Thus, purification of recombinant proteins from the cellculture of mammalian host cells may include one or more affinity (e.g.protein A) and/or ion exchange chomarographic steps.

Ion exchange chromatography is a chromatographic technique that iscommonly used for the purification of proteins. In ion exchangechromatography, charged patches on the surface of the solute areattracted by opposite charges attached to a chromatography matrix,provided the ionic strength of the surrounding buffer is low. Elution isgenerally achieved by increasing the ionic strength (i.e. conductivity)of the buffer to compete with the solute for the charged sites of theion exchange matrix. Changing the pH and thereby altering the charge ofthe solute is another way to achieve elution of the solute. The changein conductivity or pH may be gradual (gradient elution) or stepwise(step elution). In the past, these changes have been progressive; i.e.,the pH or conductivity is increased or decreased in a single direction.

For further details of the industrial purification of therapeuticantibodies see, for example, Fahmer et al., Biotechnol. Genet. Eng. Rev.18:301-27 (2001), the entire disclosure of which is expresslyincorporated by reference herein.

In addition to mammalian host cells, other eukaryotic organisms can beused as host cells for expression of the recombinant protein. Forexpression in yeast host cells, such as common baker's yeast orSaccharomyces cerevisiae, suitable vectors includeepisomally-replicating vectors based on the 2-micron plasmid,integration vectors, and yeast artificial chromosome (YAC) vectors.Other yeast suitable for recombinant production of heterologous proteinsinclude Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140(1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat.No. 4,943,529; Fleer et al., Bio/Technology, 2: 968 975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265 278 (1988)); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76: 52595263 (1979)); Schwanniomyces such as Schwanniomyces occidentalis (EP394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium (WO 91/00357 published January1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112: 284 289 (1983); Tilburn et al.,Gene, 26: 205 221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470 1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4: 475 479(1985)). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982). Expression systems forthe listed and other yeasts are well known in the art and/or arecommercially available.

For expression in insect host cells, such as Sf9 cells, suitable vectorsinclude baculoviral vectors. For expression in plant host cells,particularly dicotyledonous plant hosts, such as tobacco, suitableexpression vectors include vectors derived from the Ti plasmid ofAgrobacterium tumefaciens.

The methods of the present invention also extend to cultures ofprokaryotic host cells. Prokaryotic host cells suitable for expressingantibodies and other proteins to be protected by means of the instantinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537)and E. coli RV308 (ATCC 31,608) are also suitable. These examples areillustrative rather than limiting. Methods for constructing derivativesof any of the above-mentioned bacteria having defined genotypes areknown in the art and described in, for example, Bass et al., Proteins,8:309-314 (1990). It is generally necessary to select the appropriatebacteria taking into consideration replicability of the replicon in thecells of a bacterium. For example, E. coli, Serratia, or Salmonellaspecies can be suitably used as the host when well known plasmids suchas pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.Typically the host cell should secrete minimal amounts of proteolyticenzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Methods for the production, recovery and purification of recombinantproteins from non-mammalian host cell cultures are also well known inthe art. If the polypeptide is produced in a non-mammalian cell, e.g., amicroorganism such as fungi or E coli, the polypeptide will be recoveredinside the cell or in the periplasmic space (Kipriyanov and Little,Molecular Biotechnology, 12: 173 201 (1999); Skerra and Pluckthun,Science, 240: 1038 1040 (1988)). Hence, it is necessary to release theprotein from the cells to the extracellular medium by extraction such ascell lysis. Such disruption releases the entire contents of the cellinto the homogenate, and in addition produces subcellular fragments thatare difficult to remove due to their small size. These are generallyremoved by differential centrifugation or by filtration.

Cell lysis is typically accomplished using mechanical disruptiontechniques such as homogenization or head milling. While the protein ofinterest is generally effectively liberated, such techniques haveseveral disadvantages (Engler, Protein Purification Process Engineering.Harrison eds., 37 55 (1994)). Temperature increases, which often occurduring processing, may result in inactivation of the protein. Moreover,the resulting suspension contains a broad spectrum of contaminatingproteins, nucleic acids, and polysaccharides. Nucleic acids andpolysaccharides increase solution viscosity, potentially complicatingsubsequent processing by centrifugation, cross-flow filtration, orchromatography. Complex associations of these contaminants with theprotein of interest can complicate the purification process and resultin unacceptably low yields. Improved methods for purification ofheterologous polypeptides from microbial fermentation broth orhomogenate are described, for example, in U.S. Pat. No. 7,169,908, theentire disclosure of which is expressly incorporated herein byreference.

It is emphasized that the fermentation, recovery and purificationmethods described herein are only for illustration purposes. The methodsof the present invention can be combined with any manufacturing processdeveloped for the production, recovery and purification of recombinantproteins.

2. Antibodies

In a preferred embodiment, the methods of the present invention are usedto prevent the reduction of inter- and/or intrachain disulfide bonds ofantibodies, including therapeutic and diagnostic antibodies. Antibodieswithin the scope of the present invention include, but are not limitedto: anti-HER2 antibodies including Trastuzumab (HERCEPTIN) (Carter etal., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat. No.5,725,856); anti-CD20 antibodies such as chimeric anti-CD20 “C2B8” as inU.S. Pat. No. 5,736,137 (RITUXAN®), a chimeric or humanized variant ofthe 2H7 antibody as in U.S. Pat. No. 5,721,108B1, or Tositumomab(BEXXAR®); anti-IL-8 (St John et al., Chest, 103:932 (1993), andInternational Publication No. WO 95/23865); anti-VEGF antibodiesincluding humanized and/or affinity matured anti-VEGF antibodies such asthe humanized anti-VEGF antibody huA4.6.1 AVASTIN® (Kim et al., GrowthFactors, 7:53-64 (1992), International Publication No. WO 96/30046, andWO 98/45331, published Oct. 15, 1998); anti-PSCA antibodies(WO01/40309); anti-CD40 antibodies, including S2C6 and humanizedvariants thereof (WO00/75348); anti-CD11a (U.S. Pat. No. 5,622,700, WO98/23761, Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant etal., Transplantation 58:377-380 (1994)); anti-IgE (Presta et al., J.Immunol. 151:2623-2632 (1993), and International Publication No. WO95/19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, oras in WO 97/26912, published Jul. 31, 1997); anti-IgE (including E25,E26 and E27; U.S. Pat. No. 5,714,338, issued Feb. 3, 1998 or U.S. Pat.No. 5,091,313, issued Feb. 25, 1992, WO 93/04173 published Mar. 4, 1993,or Intentional Application No. PCT/US98/13410 filed Jun. 30, 1998, U.S.Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793 publishedNov. 19, 1998); anti-TNF-α antibodies including cA2 (REMICADE®), CDP571and MAK-195 (See. U.S. Pat. No. 5,672,347 issued Sep. 30, 1997, Lorenzet al., J. Immunol. 156(4):1646-1653 (1996), and Dhainaut et al., Crit.Care Med. 23(9):1461-1469 (1995)); anti-Tissue Factor (TF)(EuropeanPatent No. 0 420 937 B1 granted Nov. 9, 1994); anti-human α₄β₇ integrin(WO 98/06248 published Feb. 19, 1998); anti-EGFR (chimerized orhumanized 225 antibody as in WO 96/40210 published Dec. 19, 1996);anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7,1985); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and(ZENAPAX®)(See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); anti-CD4antibodies such as the cM-7412 antibody (Choy et al., Arthritis Rheum39(1):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmannet al., Nature 332:323-337 (1988)); anti-Fc receptor antibodies such asthe M22 antibody directed against FcγRI as in Graziano et al., J.Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen (CEA)antibodies such as hMN-14 (Sharkey et al., Cancer Res. 55(23Suppl):5935s-5945s (1995); antibodies directed against breast epithelial cellsincluding huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al., Cancer Res. 55(23):5852s-5856s (1995); and Richman et al., Cancer Res. 55(23 Supp):5916s-5920s (1995)); antibodies that bind to colon carcinoma cells suchas C242 (Litton et al., Eur J. Immunol. 26(1):1-9 (1996)); anti-CD38antibodies, e.g. AT 13/5 (Ellis et al., J. Immunol. 155(2):925-937(1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et al., Cancer Res55(23 Suppl):5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22antibodies such as LL2 or LymphoCide (Juweid et al., Cancer Res 55(23Suppl):5899s-5907s (1995)); anti-EpCAM antibodies such as 17-1A(PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab(REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®); anti-CMVantibodies such as PROTOVIR®; anti-HIV antibodies such as PRO542;anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR®;anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2;anti-αvβ3 antibody VITAXIN®; anti-human renal cell carcinoma antibodysuch as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-humancolorectal tumor antibody (A33); anti-human melanoma antibody R24directed against GD3 ganglioside; anti-human squamous-cell carcinoma(SF-25); and anti-human leukocyte antigen (HLA) antibodies such as SmartID10 and the anti-HLA DR antibody Oncolym (Lym-1). The preferred targetantigens for the antibody herein are: HER2 receptor, VEGF, IgE, CD20,CD11a, and CD40.

Many of these antibodies are widely used in clinical practice to treatvarious diseases, including cancer.

In certain specific embodiments, the methods of the present inventionare used for the production of the following antibodies and recombinantproteins.

Anti-CD20 Antibodies

Rituximab (RITUXAN®) is a genetically engineered chimeric murine/humanmonoclonal antibody directed against the CD20 antigen. Rituximab is theantibody called “C2B8” in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998(Anderson et al.). Rituximab is indicated for the treatment of patientswith relapsed or refractory low-grade or follicular, CD20-positive, Bcell non-Hodgkin's lymphoma. In vitro mechanism of action studies havedemonstrated that rituximab binds human complement and lyses lymphoid Bcell lines through complement-dependent cytotoxicity (CDC) (Reff et al.,Blood 83(2):435-445 (1994)). Additionally, it has significant activityin assays for antibody-dependent cellular cytotoxicity (ADCC). Morerecently, rituximab has been shown to have anti-proliferative effects intritiated thymidine incorporation assays and to induce apoptosisdirectly, while other anti-CD19 and CD20 antibodies do not (Maloney etal., Blood 88(10):637a (1996)). Synergy between rituximab andchemotherapies and toxins has also been observed experimentally. Inparticular, rituximab, sensitizes drug-resistant human B cell lymphomacell lines to the cytotoxic effects of doxorubicin, CDDP, VP-1 6,diphtheria toxin and ricin (Demidem et al., Cancer Chemotherapy &Radiopharmaceuticals 12(3):177-186 (1997)). In vivo preclinical studieshave shown that rituximab depletes B cells from the peripheral blood,lymph nodes, and bone marrow of cynomolgus monkeys, presumably throughcomplement and cell-mediated processes (Reff et al., Blood 83(2):435-445(1994)).

Patents and patent publications concerning CD20 antibodies include U.S.Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and 5,843,439, as well asU.S. patent application Nos. US 2002/0197255A1, US 2003/0021781A1, US2003/0082172 A1, US 2003/0095963 A1, US 2003/0147885 A1 (Anderson etal.); U.S. Pat. No. 6,455,043B1 and WO0009160 (Grillo-Lopez, A.);WO00/27428 (Grillo-Lopez and White); WO00/27433 (Grillo-Lopez andLeonard); WO00/44788 (Braslawsky et al.); WO01/10462 (Rastetter, W.);WO01/10461 (Rastetter and White); WO01/10460 (White and Grillo-Lopez);U.S. application No. US2002/0006404 and WO2/04021 (Hanna and Hariharan);U.S. application No. US2002/0012665 A1 and WO01/74388 (Hanna, N.); U.S.application No. US 2002/0058029 A1 (Hanna, N.); U.S. application No. US2003/0103971 A1 (Hariharan and Hanna); U.S. application No.US2002/0009444A1, and WO01/80884 (Grillo-Lopez, A.); WO01/97858 (White,C.); U.S. application No. US2002/0128488A1 and WO02/34790 (Reff, M.);W002/060955 (Braslawsky et aL); WO2/096948 (Braslawsky et al.);WO02/079255 (Reff and Davies); U.S. Pat. No. 6,171,586B1, and WO98/56418(Lam et al.,); WO98/58964 (Raju, S.); WO99/22764 (Raju, S.); WO99/51642,U.S. Pat. Nos. 6,194,551B, 6,242,195B1, 6,528,624B1 and 6,538,124(Idusogie et al.); WO00/42072 (Presta, L.); WO00/67796 (Curd et al.);WO01/03734 (Grillo-Lopez et al.); U.S. application No. US 2002/0004587A1and WO01/77342 (Miller and Presta); U.S. application No. US2002/0197256(Grewal, I.); U.S. application No. US 2003/0157108 A1 (Presta, L.); U.S.Pat. Nos. 6,090,365B1, 6,287,537B1, 6,015,542, 5,843,398, and 5,595,721,(Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180, 5,721,108, and6,120,767 (Robinson et al.); U.S. Pat. No. 6,410,391B1 (Raubitschek etal.); U.S. Pat. No. 6,224,866B1 and WO00/20864 (Barbera-Guillem, E.);WO01/13945 (Barbera-Guillem, E.); WO00/67795 (Goldenberg); U.S.application No. US 2003/01339301 A1 and WO00/74718 (Goldenberg andHansen); WO00/76542 (Golay et al.); WO01/72333 (Wolin and Rosenblatt);U.S. Pat. No. 6,368,596B1 (Ghetie et al.); U.S. application No.US2002/0041847 A1, (Goldenberg, D.); U.S. application No.US2003/0026801A1 (Weiner and Hartmann); WO02/102312 (Engleman, E.); U.S.patent application No. 2003/0068664 (Albitar et al.); WO03/002607(Leung, S.); WO 03/049694 and US 2003/0185796 A1 (Wolin et aL)WO03/061694 (Sing and Siegall); US 2003/0219818 A1 (Bohen et al.); US2003/0219433 A1 and WO 03/068821 (Hansen et aL) each of which isexpressly incorporated herein by reference. See, also, U.S. Pat. No.5,849,898 and EP application no. 330,191 (Seed et aL); U.S. Pat. No.4,861,579 and EP332,865A2 (Meyer and Weiss); U.S. Pat. No. 4,861,579(Meyer et al.) and WO95/03770 (Bhat et al.).

Publications concerning therapy with Rituximab include: Perotta andAbuel “Response of chronic relapsing ITP of 10 years duration toRituximab” Abstract #3360 Blood 10(1)(part 1-2): p. 88B (1998); Stashiet al., “Rituximab chimeric anti-CD20 monoclonal antibody treatment foradults with chronic idopathic thrombocytopenic purpura” Blood98(4):952-957 (2001); Matthews, R. “Medical Heretics” New Scientist (7Apr. 2001); Leandro et al., “Clinical outcome in 22 patients withrheumatoid arthritis treated with B lymphocyte depletion” Ann Rheum Dis61:833-888 (2002); Leandro et al., “Lymphocyte depletion in rheumatoidarthritis: early evidence for safety, efficacy and dose response.Arthritis & Rheumatism 44(9): S370 (2001); Leandro et al., “An openstudy of B lymphocyte depletion in systemic lupus erythematosus”,Arthritis & Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge“Sustained improvement in rheumatoid arthritis following a protocoldesigned to deplete B lymphocytes” Rheumatology 40:205-211 (2001);Edwards et al., “B-lymphocyte depletion therapy in rheumatoid arthritisand other autoimmune disorders” Biochem. Soc. Trans. 30(4):824-828(2002); Edwards et al., “Efficacy and safety of Rituximab, a B-celltargeted chimeric monoclonal antibody: A randomized, placebo controlledtrial in patients with rheumatoid arthritis. Arthritis & Rheumatism46(9): S197 (2002); Levine and Pestronk “IgM antibody-relatedpolyneuropathies: B-cell depletion chemotherapy using Rituximab”Neurology 52: 1701-1704 (1999); DeVita et al., “Efficacy of selective Bcell blockade in the treatment of rheumatoid arthritis” Arthritis &Rheumatism 46:2029-2033 (2002); Hidashida et al., “Treatment ofDMARD-Refractory rheumatoid arthritis with rituximab.” Presented at theAnnual Scientific Meeting of the American College of Rheumatology;October 24-29; New Orleans, La. 2002; Tuscano, J. “Successful treatmentof Infliximab-refractory rheumatoid arthritis with rituximab” Presentedat the Annual Scientific Meeting of the American College ofRheumatology; October 24-29; New Orleans, La. 2002. Sarwal et al, N.Eng. J. Med. 349(2):125-138 (Jul. 10, 2003) reports molecularheterogeneity in acute renal allograft rejection identified by DNAmicroarray profiling.

In various embodiments, the invention provides pharmaceuticalcompositions comprising humanized 2H7 anti-CD20 antibodies. In specificembodiments, the humanized 2H7 antibody is an antibody listed in Table2.

TABLE 2 Humanized anti-CD20 Antibody and Variants Thereof V_(L) V_(H)Full L chain Full H chain 2H7 SEQ ID SEQ ID SEQ ID SEQ ID variant NO.NO. NO. NO. A 1 2 6 7 B 1 2 6 8 C 3 4 9 10 D 3 4 9 11 F 3 4 9 12 G 3 4 913 H 3 5 9 14 I 1 2 6 15

Each of the antibody variants A, Band I of Table 2 comprises the lightchain variable sequence (V_(L)):

(SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG TKVEIKR;and

the heavy chain variable sequence (V_(H)):

(SEQ ID NO: 2) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS.

Each of the antibody variants C, D, F and G of Table 2 comprises thelight chain variable sequence (V_(L)):

(SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTK VEIKR;and

the heavy chain variable sequence (V_(H)):

(SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS.

The antibody variant H of Table 2 comprises the light chain variablesequence (V_(L)) of SEQ ID NO:3 (above) and the heavy chain variablesequence (V_(H)):

(SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SYRYWYFDVWGQGTLVTVSS

Each of the antibody variants A, Band I of Table 2 comprises the fulllength light chain sequence:

(SEQ ID NO: 6) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC.

Variant A of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 7) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variant B of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variant I of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Each of the antibody variants C, D, F, G and H of Table 2 comprises thefull length light chain sequence:

(SEQ ID NO: 9) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC.

Variant C of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variant D of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 11) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variant F of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 12) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variant G of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHWHYTQKSLSLSPGK.

Variant H of Table 2 comprises the full length heavy chain sequence:

(SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In certain embodiments, the humanized 2H7 antibody of the inventionfurther comprises amino acid alterations in the IgG Fc and exhibitsincreased binding affinity for human FcRn over an antibody havingwild-type IgG Fc, by at least 60 fold, at least 70 fold, at least 80fold, more preferably at least 100 fold, preferably at least 125 fold,even more preferably at least 150 fold to about 170 fold.

The N-glycosylation site in IgG is at Asn297 in the C_(H)2 domain.Humanized 2H7 antibody compositions of the present invention includecompositions of any of the preceding humanized 2H7 antibodies having aFc region, wherein about 80-100% (and preferably about 90-99%) of theantibody in the composition comprises a mature core carbohydratestructure which lacks fucose, attached to the Fc region of theglycoprotein. Such compositions were demonstrated herein to exhibit asurprising improvement in binding to Fc(RIIIA (F158), which is not aseffective as Fc(RIIIA (V158) in interacting with human IgG. Fc(RIIIA(F158) is more common than Fc(RIIIA (V158) in normal, healthy AfricanAmericans and Caucasians. See Lehrnbecher et al., Blood 94:4220 (1999).Historically, antibodies produced in Chinese Hamster Ovary Cells (CHO),one of the most commonly used industrial hosts, contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 and Lec13, however, canproduce antibodies with 78 to 98% nonfucosylated species. Shinkawa etal., J Bio. Chem. 278 (5), 3466-347 (2003), reported that antibodiesproduced in YB2/0 and Lec13 cells, which have less FUT8 activity, showsignificantly increased ADCC activity in vitro. The production ofantibodies with reduced fucose content are also described in e.g., Li etal., (GycoFi) “Optimization of humanized IgGs in glycoengineered Pichiapastoris” in Nature Biology online publication 22 Jan. 2006; Niwa R. etal., Cancer Res. 64(6):2127-2133 (2004); US 2003/0157108 (Presta); U.S.Pat. No. 6,602,684 and US 2003/0175884 (Glycart Biotechnology); US2004/0093621, US 2004/0110704, US 2004/0132140 (all of Kyowa HakkoKogyo).

A bispecific humanized 2H7 antibody encompasses an antibody wherein onearm of the antibody has at least the antigen binding region of the Hand/or L chain of a humanized 2H7 antibody of the invention, and theother arm has V region binding specificity for a second antigen. Inspecific embodiments, the second antigen is selected from the groupconsisting of CD3, CD64, CD32A, CD16, NKG2D or other NK activatingligands.

Anti-HER2 Antibodies

A recombinant humanized version of the murine HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCFPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anticancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumabreceived marketing approval from the Food and Drug Administration (FDA)Sep. 25, 1998 for the treatment of patients with metastatic breastcancer whose tumors overexpress the HER2 protein. In November 2006, theFDA approved Herceptin as part of a treatment regimen containingdoxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatmentof patients with HER2-positive, node-positive breast cancer.

In one embodiment, the anti-HER2 antibody comprises the following V_(L)and V_(H) domain sequences:

humanized 2C4 version 574 antibody V_(L) (SEQ ID NO:16)

DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGT KVEIK.

and humanized 2C4 version 574 antibody V_(H) (SEQ ID NO:17)

EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGP SFYFDYWGQGTLVTVSS.

In another embodiment, the anti-HER2 antibody comprises the V_(L) (SEQID NO:18) and V_(H) (SEQ ID NO:19) domain sequences of trastuzumab asshown in FIG. 21 and FIG. 22, respectively.

Other HER2 antibodies with various properties have been described inTagliabue et al., Int. J. Cancer 47:933-937 (1991); McKenzie et al.,Onogene 4:543-548 (1989); Maier et al., Cancer Res. 51:5361-5369 (1991);Bacus et al., Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal., PNAS (USA) 88:8691-8695 (1991); Bacus et al., Cancer Research52:2580-2589 (1992); Xu et al., Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al., Cancer Research 52:2771-2776 (1992);Hancock et al., Cancer Res 51:4575-480 (1991); Shawyer et al., CancerRes. 54:1367-1373 (1994); Arteaga et al., Cancer Res. 54:3758-3765(1994); Harwerth et al., J. Biol. Chem. 267:15160-15167 (1992); U.S.Pat. No. 5,783,186; and Kapper et al., Oncogene 14:2099-2109 (1997).

Anti-VEGF Antibodies

The anti-VEGF antibodies may, for example, comprise the followingsequences:

In one embodiment, the anti-VEGF antibody comprises the following V_(L)sequence (SEQ ID NO:20):

DIQMTQTTSS LSASLGDRVI ISCSASQDIS NYLNWYQQKPDGTVKVLIYF TSSLHSGVPS RFSGSGSGTD YSLTISNLEPEDIATYYCQQ YSTVPWTFGG GTKLEIKR;and

the following V_(H) sequence (SEQ ID NO:21):

EIQLVQSGPE LKQPGETVRI SCKASGYTFT NYGMNWVKQAPGKGLKWMGW INTYTGEPTY AADFKRRFTF SLETSASTAYLQISNLKNDD TATYFCAKYP HYYGSSHWYF DVWGAGTTVT VSS.

In another embodiment, the anti-VEGF antibody comprises the followingV_(L) sequence (SEQ ID NO:22):

DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKPGKAPKVLIYF TSSLHSGVPS RFSGSGSCTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR;andthe following V_(H) sequence (SEQ ID NO:23):

EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQAPGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS.

In a third embodiment, the anti-VEGF antibody comprises the followingV_(L) sequence (SEQ ID NO:24):

DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKPGKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR;and

the following V_(H) sequence (SEQ ID NO:25):

EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQAPGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSS.

Anti-CD11a Andibodies

The humanized anti-CD11a antibody efalizumab or Raptiva® (U.S. Pat. No.6,037,454) received marketing approval from the Food and DrugAdministration on Oct. 27, 2003 for the treatment for the treatment ofpsoriasis. One embodiment provides for anti-human CD111a antibodycomprising the V_(L) and V_(H) sequences of HuMHM24 below:

V_(L) (SEQ ID NO: 26 ):DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGT KVEIKR; andV_(H) (SEQ ID NO: 27):EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGMIHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGIYF YGTTYFDYWGQGTLVTVSS

The anti-human CD11a antibody may comprise the V_(H) of SEQ ID NO:27 andthe full length L chain of HuMHM24 having the sequence of:

(SEQ ID NO: 28) DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC,orthe L chain above with the H chain having the sequence of:

(SEQ ID NO: 29) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGMIHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGIYFYGTTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.

Antibodies to the DR5 receptor (anti-DR5) antibodies can also beproduced in accordance with the present invention. Such anti-DR5antibodies specifically include all antibody variants disclosed in PCTPublication No. WO 20061083971, such as the anti-DR5 antibodiesdesignated Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3,7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2,9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and 25.3, especiallyApomab 8.3 and Apomab 7.3, preferably Apomab 7.3. The entire content ofWO 2006/083971 is hereby expressly incorporated by reference.

3. Other Disulfide-Containing Proteins

In addition to antibodies, the methods of the present invention findutility in the manufacturing of other polypeptides including disulfidebonds. Representative examples of such polypeptides include, withoutlimitation, the following therapeutic proteins: tissue plasminogenactivators (t-PAs), such as human tissue plasminogen activator (htPA,alteplase, ACTIVASE®), a thrombolytic agent for the treatment ofmyocardial infarction; a TNKase™, a ht-PA variant with extendedhalf-life and fibrin specificity for single-bolus administration;recombinant human growth hormone (rhGH, somatropin, NUTROPIN®,PROTROPIN®) for the treatment of growth hormone deficiency in childrenand adults; and recombinant human deoxyribonuclease I (DNase I) for thetreatment of cystic fibrosis (CF).

Examples of disulfide-containing biologically important proteins includegrowth hormone, including human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factors such as factor VIIIC,factor IX, tissue factor, and von Willebrands factor, anti-clottingfactors such as Protein C: atrial natriuretic factor, lung surfactant; aplasminogen activator, such as urokinase or human urine or tissue-typeplasminogen activator (t-PA); bombesin; thrombin; hemopoietic growthfactor, tumor necrosis factor-alpha and -beta; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); a serum albumin such ashuman serum albumin; Muellerian-inhibiting substance; relaxin A-chain;relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; amicrobial protein, such as beta-lactamase; DNase; IgE; a cytotoxicT-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;activin; vascular endothelial growth factor (VEGF); receptors forhormones or growth factors; Protein A or D; rheumatoid factors; aneurotrophic factor such as bone-derived neurotrophic factor (BDNF),neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nervegrowth factor such as NGF-s; platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-1),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; osteoinductivefactors; immunotoxins; a bone morphogenetic protein (BMP); an interferonsuch as interferon-alpha, -beta, and -gamma; colony stimulating factors(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (Is), e.g., IL-1 toIL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor, viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA4 and VCAM; a tumor associated antigen such as HER2,HER3 or HER4 receptor, and fragments of any of the above-listedpolypeptides.

4. General Methods for the Recombinant Production of Antibodies

The antibodies and other recombinant proteins herein can be produced bywell known techniques of recombinant DNA technology. Thus, aside fromthe antibodies specifically identified above, the skilled practitionercould generate antibodies directed against an antigen of interest, e.g.,using the techniques described below.

Antigen Selection and Preparation

The antibody herein is directed against an antigen of interest.Preferably, the antigen is a biologically important polypeptide andadministration of the antibody to a mammal suffering from a disease ordisorder can result in a therapeutic benefit in that mammal. However,antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated. Where the antigen is a polypeptide, it may be atransmembrane molecule (e.g. receptor) or ligand such as a growthfactor. Exemplary antigens include those proteins described in section(3) below. Exemplary molecular targets for antibodies encompassed by thepresent invention include CD proteins such as CD3, CD4, CD8, CD19, CD20,CD22, CD34, CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor, cell adhesion molecules such asLFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin includingeither α or β subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; IgE; blood group antigens;flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; proteinC, or any of the other antigens mentioned herein. Antigens to which theantibodies listed above bind are specifically included within the scopeherein.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule.

Other antigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the antigen to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of antigen or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, Protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. Preferably theProtein A chromatography procedure described herein is used.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad Sci. USA, 81:6851 (1984)), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In a further embodiment, monoclonal antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalhybridoma techniques for isolation of monoclonal antibodies.

Humanized and Human Antibodies

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman FR for the humanized antibody (Sims et al., J. Immunol., 151:2296(1993)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Nat. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and Duchosal et al., Nature 355:258(1992). Human antibodies can also be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech14:309 (1996)).

Antibody Fragments

Various techniques have been developed for the production of antibodyfragments.

Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992) and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv) (see WO93/16155).

Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the C_(H)3domain of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V andV_(H) domains of another fragment, thereby forming two antigen-bindingsites. Another strategy for making bispecific antibody fragments by theuse of single-chain Fv (sFv) dimers has also been reported. See Gruberet al., J. Immunol., 152:5368 (1994). Alternatively, the antibodies canbe “linear antibodies” as described in Zapata et al., Protein Eng.8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)I-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991).

Immunoadhesins

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the extracellular domain (ECD) ofa receptor) with the hinge and Fc regions of an immunoglobulin heavychain. Ordinarily, when preparing the immunoadhesins of the presentinvention, nucleic acid encoding the binding domain of the adhesin willbe fused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least, functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H3) domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG.IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

-   -   AC_(L)-AC_(L);    -   AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H));    -   AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),        V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H))    -   AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), or        V_(L)C_(L)-AC_(H));    -   V_(L)C_(L)-AC_(H)-(AC-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and    -   (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom, etal., Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1

Description of Materials and Methods

The following materials and methods were used in Examples 2-8 below.

Materials

Materials and devices used in the experiments described in theexperimental examples include: stainless steel vials (mini-tanks, FlowComponents, Dublin, Calif.; short (50 cc) and tall (55 cc)); dialysistubing (Spectra/Por, 6-8000 MWCO, cat. #132645), 0.22 μm filter(Millipore Millipak Gamma Gold cat. # MPGL04GH2); phosphate bufferedsaline (PBS, EMD, cat. #6506); ethylenediaminetetraacetic acid (EDTA,Sigma, cat. # E4884); α-nicotinamide adenine dinucleotide phosphate(NADPH, Calbiochem, cat. #481973); dehydroepiandrosterone (DHEA, TCI,cat. # D0044); cupric sulfate (Sigma, cat. # C8027), glucose-6-phosphate(G6P, Calbiochem, cat. #346764); aurothioglucose (ATG, USP, cat.#1045508); aurothiomalate (ATM, Alfa Aesar, cat. #39740); reducedglutathione (GSH, J. T. Baker, cat. # M770-01); monobromobimane (mBB,Fluka, cat. #69898); histidine (J. T. Baker, cat. #2080-05); sodiumsulfate (J. T. Baker, cat. #3897-05); Trx (Sigma, cat. # T8690); TrxR(Sigma, cat. # T9698). All chemicals and reagents were used as receivedwith no further purification. Stock solutions of EDTA (250 mM, pH 7.5),CuSO₄ (10 mM), ATG (30 mM), ATM (30 mM), NADPH (75 mM), G6P (300 mM)were prepared for use in the mini-tank time course studies.

Generation of Cell Culture Fluid (CCF)

In order to generate ocrelizumab CCF for the various reduction studies,a representative small-scale fermentation process was utilized similarto the methods described previously (Chaderjian et al., 2005). Briefly,3 liter glass stirred-tank Applikon® bioreactors fitted with pitchedblade impellers were used for the inoculum-train and production cultureswith the ocrelizumab media components. The bioreactors were outfittedwith calibrated dissolved oxygen (DO), pH and temperature probes. DO,pH, temperature, and agitation rate were controlled using digitalcontrol units to the defined parameters of the ocrelizumab manufacturingprocess. The working volume for both the inoculum-train and productioncultures was 1.5 L. Daily samples were analyzed on a NOVA Bioprofileblood gas analyzer to ensure the accuracy of the on-line value for pHand dissolved oxygen as well as to monitor the glucose, lactate,ammonium, glutamine, glutamate, and sodium concentrations in thecultures. Daily samples were also taken to monitor cell growth,viability, and titer. Cell growth was measured both by viable cellcounts using a ViCell as well as on a packed cell volume (PCV) basis.Culture viability was determined by trypan blue exclusion on a ViCellinstrument. Supernatant samples were assayed by an HPLC-based method tomeasure ocrelizumab titer values.

Harvested Cell Culture Fluid (HCCF) Preparation

Complete lysis of CCF was achieved by high pressure homogenization usinga Microfluidics HC-8000 homogenizer. The pressure regulator of theinstrument was set to 4,000-8,000 psi, and the CCF was pulled in throughthe homogenizer to obtain complete cell lysis (membrane breakage) aftera single pass. The CCF homogenate was collected once water was purgedthrough the system. The homogenate was transferred to centrifuge bottlesand centrifuged in a Sorval RC-3B rotor centrifuge at 4,500 rpm for 30minutes at 20° C. The centrate was decanted and then depth filteredfollowed by 0.22 μm sterile filtration using a peristaltic pump withsilicon tubing to generate the final HCCF from the homogenized CCF (100%cell lysis). Alternatively, the CCF was centrifuged straight from thefermentor without any homogenization and then the centrate was filteredwith a sterile 0.22 μm filter to generate the HCCF.

Mini-Tank Handling

A laminar flow hood was used in handling all mini-tanks and allmaterials used in the HCCF incubation experiments were either autoclavedor rinsed using 70% isopropanol to minimize bacterial contamination.

Lactate Dehydrogenase Assay

For lactate dehydrogenase assay, see Babson & Babson (1973) and Legrandet al., (1992), which are hereby incorporated by reference.

Dialysis Experiment

A dialysis experiment was carried out in order to determine whether thecomponents causing reduction of ocrelizumab were small molecules ormacromolecules (i.e. enzymes). A sample of 3 mL of purified andformulated ocrelizumab (30.2 mg/mL) was dialyzed against 1 L ofphosphate buffered saline (PBS, 10 mM pH 7.2) for 24 hours and the PBSwas changed after 8 hours. The concentration of the ocrelizumab samplewas then adjusted to 1 mg/mL using the absorbance at 280 nm. Aliquotswere stored at −70° C. prior to use. Dialysis tubing was hydratedovernight in a 0.05% azide solution and rinsed with sterile water priorto use. The HCCF obtained from homogenization of CCF from a 3-Lfermentor was thawed and filtered through a 0.22 μm Millipak filterusing a peristaltic pump. Six short mini-tanks were filled with 30 mL ofHCCF each. To each mini-tank, 500 μL of ocrelizumab sample in sealeddialysis tubing was added. The mini-tanks were sealed and loaded into abench top mixer (Barnstead Lab-Line MAX Q 4000) operating at 35 rpm andambient temperature. For each time-point, one mini-tank was removed fromthe mixer, and aliquots of the HCCF (in the mini-tank) and ocrelizumabsample (in the dialysis bag) were taken and stored at −70° C. untilanalyzed with the free thiol assay and the Bioanalyzer assay (describedbelow).

Test Inhibitors for Reduction in a Small-Scale In Vitro System

A tall mini-tank was filled with 27 mL of HCCF. Depending on theexperiment design, various reagents (NADPH, G6P, inhibitors of G6PD orTrxR) were added to the desired concentration, and the final volume inthe mini-tank was brought to 30 mL with PBS (10 mM pH 7.2). Themini-tanks were sealed and loaded into a bench top mixer running at 35rpm and ambient temperature. At each-time point for sampling, theexteriors of the mini-tanks were sterilized with 70% IPA and opened in alaminar flow hood for the removal of an aliquot. The mini-tanks werethen re-sealed and loaded back into the bench top mixer. All aliquotswere stored at −70° C. until analyzed with the free thiol assay andBioanalyzer assay (described below).

In Vitro Trx/TrxR Reductase Studies

A commercial TrxR (rat liver) solution (4 μM) was diluted with water toyield a 2.86 μM solution. Lyophilized Trx (human) was reconstituted withPBS (10 mM, pH 7.2) yielding a 500 μM solution. A solution of 20 mMNADPH and 10 mM ATG and ATM solutions were prepared in water.

In a black polypropylene 1.5 mL micro centrifuge tube, 437 μL PBS, 25 μLNADPH, 16 μL formulated ocrelizumab solution (30.2 mg/mL) and 5 μL Trxwere gently mixed. The reaction was initiated by the addition of 17.5 μLTrxR. The reaction was incubated at room temperature for 24 hours.Aliquots of 20 μL were taken at each sampling time-point and stored at−70° C. until analyzed by the Bioanalyzer assay (see below). Controlswere performed to determine if the enzymatic pathway was active when anenzyme was omitted by substituting an equal volume of PBS for either Trxand/or TrxR in the reaction mixture.

Inhibition of the Trx system was demonstrated using the same reactionconditions described above with the addition of 5 μL ATG or ATM. Todemonstrate the inhibition of Trx system by Cu²⁺, 2.5 μL of CuSO₄ (10mM) was added to reaction mixture using the same enzymes but a differentbuffer (10 mM histidine, 10 mM Na₂SO₄, 137 mM NaCl, 2.5 mM KCl, pH 7.0)to prevent formation of insoluble Cu₃(PO₄)₂.

Free Thiol Assay

A standard curve using GSH was generated in PBS (10 mM, pH 6.0±0.05).From a 110-mM GSH solution, standards were prepared at concentrations of0, 5.5, 11, 22, 44, 55, 110 and 550 μM through serial dilution. From anacetonitrile stock solution of mBB (10 mM stored at −20° C.), a 100 μMsolution of mBB was prepared in PBS (10 mM, pH 10.00.05) and stored awayfrom light.

In a black, flat bottomed 96 well plate, 100 μL of mBB was dispensedinto each well. For the standard curve, 10 μL of standard GSH solutionwas added yielding a working pH of 8.0±0.2. For samples, 10 μL of samplewas added to the wells. All wells were prepared in triplicate. The platewas incubated at room temperature for 1 hour in the dark then read usinga fluorescence plate reader (Molecular Devices SpectraMax® Gemini XS)with an excitation wavelength of 390 nm and an emission wavelength of490 nm. A linear standard curve was generated using the average resultof the three standard wells plotted versus GSH concentration. Free thiollevels in samples were calculated from the linear equation of thestandard curve using the average value of the three sample wells.

Bioanalyzer Assay

Capillary electrophoresis measurements were acquired using the Agilent2100 Bioanalyzer. Sample preparation was carried out as described in theAgilent Protein 230 Assay Protocol (manual part number G2938-90052) withminor changes. HCCF samples were diluted, 1:4 and Protein A samples werediluted to 1.0 g/L with water prior to preparation. For HCCF samples atthe denaturing step, 24 μL of a 50 mM iodoacetamide (IAM), 0.5% SDSsolution was added in addition to the 2 μL of denaturing solutionprovided. For Protein A samples, 0.5% SDS with no IAM and 2 μL ofdenaturing solution were used. Digital gel-like images were generatedusing Agilent 2100 Expert software.

Stock Solutions for HCCF Hold Time Studies

Three separate stock solutions were used in the lab scale HCCF hold timestudies: (1) 250 mM stock solution of EDTA (pH 7.4) prepared using EDTA,disodium dihydrate (Mallinckrodt, cat. #7727-06 or Sigma, cat. # E-5134)and EDTA, tetrasodium dihydrate (Sigma, cat. # E-6511), (2) 50 mM stocksolution of cupric sulfate pentahydrate (CuSO₄, Sigma, cat. # C-8027),and (3) 1 M acetic acid solution (Mallinckrodt, cat. # V193).

Inhibitor Additions and Cell Culture Fluid (CCF) Blending

A stock solution of either 250 mM EDTA or 50 mM CuSO₄ was added to theCCF prior to homogenization to evaluate a range of final concentrationsto prevent antibody disulfide reduction. Once the final HCCF wasgenerated from the homogenized CCF, these solutions were then mixed withthe HCCF generated from the non-homogenized CCF (also containing EDTA orCuSO₄) in order to dilute and decrease the total level of cell lysis tobelow the 100% maximum. Alternatively, a stock solution of 1 M aceticacid was added to a final blended HCCF solution (homogenized CCF andnon-homogenized CCF) to decrease the pH of the solution to preventantibody disulfide reduction.

Approximately 30-50 mL of each HCCF solution (containing EDTA, CuSO₄,acetic acid, or no addition for the control) was held in a 50 mL 316Lstainless steel vial. The vial was sealed with a clamp, and the solutionwas not aerated or agitated. The vial was stored at room temperature(18-22° C.). At pre-determined time points, the solution was removed andpurified over a lab scale protein A affinity resin.

Similar results can be obtained with other oxidizing agents, such as,for example, cystine and oxidized glutathione.

Air Sparging

To evaluate air sparging of the HCCF generated from homogenized CCF toprevent antibody disulfide reduction, 3-L glass or 15-L stainless steelvessels were utilized. Approximately 1-5 L of HCCF was 0.22 μm sterilefiltered into each sterilized vessel. Experimental conditions weremaintained at 18-22° C. and 50 (15-L fermentor) or 275 rpm (3-Lfermentor) agitation either with or without pH control by the additionof carbon dioxide. Solutions were either sparged with air to increasethe dissolved oxygen level to air saturation or with nitrogen (control)to remove any dissolved oxygen in solution. Gas flow to each vessel wasvariable dependent upon whether a constant aeration rate was used or aminimum level of dissolved oxygen was maintained. At pre-determined timepoints, 25-50 mL samples were removed from both vessels and purifiedover a lab scale protein A affinity resin prior to analysis.

Protein A Processing

Antibody in harvested cell culture fluid samples can be captured andpurified using a specific affinity chromatography resin. Protein A resin(Millipore, Prosep-vA High Capacity) was selected as the affinity resinfor antibody purification. The resin was packed in a 0.66 cm innerdiameter glass column (Omnifit®) with a 14 cm bed height resulting in a4.8 mL final column volume. Chromatography was performed using an AKTAExplorer 100 chromatography system (GE Healthcare).

The resin was exposed to buffers and HCCF at a linear flow rate between350-560 cm/hr. The resin was equilibrated with 25 mM Tris, 25 mM NaCl, 5mM EDTA. pH 7.1. For each purification, the resin was loaded between5-15 mg antibody per mL of resin. The antibody concentration in the HCCFwas determined using an immobilized protein A HPLC column (AppliedBiosystems, POROS A). After loading, the resin was washed with 25 mMTris, 25 mM NaCl, 5 mM EDTA, 0.5 M TMAC, pH 7.1, and then the antibodywas eluted using 0.1M acetic acid, pH 2.9. Elution pooling was based onUV absorbance at 280 nm measured inline after the column. The purifiedelution pools were pH-adjusted using 1 M Sodium HEPES to pH 5.0-5.5.After regeneration of the resin with 0.1M phosphoric acid, the same orsimilar packed resins were used for subsequent purification of otherHCCF solutions.

The antibody concentration in the purified protein A pool was measuredusing UV spectrometry at 280 nm. The purified protein A elution poolswere analyzed by the Bioanalyzer assay to quantitate the percentage ofintact antibody at 150 kDa molecular weight.

Example 2

Dialysis Experiment

A dialysis experiment was designed and carried out to determine if thereduction of ocrelizumab was caused by small reducing molecules ormacromolecules (e.g., enzymes). In this dialysis experiment, purifiedintact ocrelizumab was placed in a dialysis bag with a molecular weightcut off (MWCO) of 7000 and incubated the dialysis bag in HCCF containingocrelizumab in a stainless steel mini-tank. As shown in FIGS. 1 and 2,the ocrelizumab inside the bag was not reduced after the incubationperiod (FIG. 1), whereas the ocrelizumab outside the bag in the HCCF wassignificantly reduced soon after the incubation started. This wasevidenced by the loss of intact ocrelizumab (˜150 kDa) and the formationof ocrelizumab fragments (various combinations of heavy and lightchains)(FIG. 2). The mass spectrometry analysis of the ocrelizumab inthe protein A elution pools from the reduced manufacturing runsindicated that those observed fragments were formed by reduction of onlythe inter-chain disulfide bonds.

The free thiol measurement showed that no free thiols were presentinside the dialysis bag at the beginning of the incubation; however thelevels of free thiols inside and outside the dialysis bag becomecomparable in less than five hours after the incubation started,indicating that the small molecule components in the HCCF are fullyequilibrated inside and outside the dialysis bag (FIG. 3). Since thereduction was observed only outside but not inside the dialysis bag witha MWCO of 7000 Da, the molecular weight of the reducing molecule(s) mustbe greater than 7000 Da. Thus, an enzymatic reaction is responsible forthe reduction of ocrelizumab.

Example 3

Reduction of Ocrelizumab (rhuMAb 2H7. Variant A) by Trx/TrxR In Vitro

The Trx system was tested for its ability to reduce ocrelizumab in vitroby incubating intact ocrelizumab with Trx, TrxR, and NADPH. TheBioanalyzer results indicate that ocrelizumab was reduced in vitro bythe Trx system (FIG. 5). The rate of reduction in this in vitro systemappears to be slower than that in the HCCF (for example when compared tothe reduction shown in FIG. 2). This is likely due to lowerconcentrations of the enzymes (Trx and Trx-R) and/or the buffer systemused in the in vitro reaction because reaction rate of Trx system isdependent on both the enzyme concentrations and buffer systems.

Example 4

Inhibitors of the Trx System

(i) Inhibition of Reduction of Recombinant Antibody by Cupric Sulfate

Cupric sulfate is known for its ability to provide oxidizing redoxpotential and has been used in the cell culture processes to minimizefree thiol (i.e., minimize unpaired cysteine) levels in recombinantantibody molecules (Chaderjian et al., 2005, supra). Cupric sulfate wastested for efficacy in inhibiting the Trx system in vitro and thesubsequent reduction of ocrelizumab. In this in vitro reductionexperiment, the buffer system was changed from PBS to histidine sulfateto avoid the formation of insoluble Cu₃(PO₄)₂. FIG. 8 shows thatocrelizumab was readily reduced by the Trx system in the histidinesulfate buffer (even faster than in PBS buffer). The addition of CuSO₄to this reaction clearly inhibits the ocrelizumab reduction (FIG. 9).

(ii) Inhibition of Reduction of Recombinant Antibody in HCCF by ATG andATM

Two commercially available specific inhibitors of TrxR, aurothioglucose(ATG) and aurothiomalate (ATM), were tested for their ability to inhibitthe Trx system in vitro and the reduction of ocrelizumab. Both ATG andATM can effectively inhibit the reduction of ocrelizumab in the assaydescribed above (see FIGS. 6 and 7). The addition of aurothioglucose oraurothiomalate, at a concentration of 1 mM to the same reaction mixtureas described in the caption for FIG. 5 effectively inhibited theocrelizumab reduction as shown in the digital gel-like image fromBioanalyzer analysis.

If the Trx system was active in the HCCF and reduced ocrelizumab asobserved in the manufacturing runs resulting in reduced antibodymolecules or in the lab scale experiments, both gold compounds (ATG andATM) should be able to inhibit the reduction of ocrelizumab in HCCF.FIG. 10 shows that ocrelizumab was readily reduced in an HCCF fromhomogenized CCT generated from a 3-L fermentor after a period ofincubation. However, the ocrelizumab reduction event was completelyinhibited when either 1 mM ATG or ATM was added to the HCCF (FIGS. 11and 12). These results demonstrated that the Trx system is active in theHCCF and is directly responsible for the reduction of ocrelizumab.

Example 5

The Source of NADPH for Trx System Activity and the Roles of G6P andGlucose in Reduction Mechanism

The reduction of disulfides by the Trx system requires the reducingequivalents from NADPH (FIG. 4). The main cellular metabolic pathwaythat provides NADPH for all reductive biosynthesis reactions is thepentose phosphate pathway. For the antibody reduction event to occur,the enzymes in this pathway must be still active in the HCCF in order tokeep the Trx system active. At a minimum, the first step in the pentosephosphate pathway (catalyzed by G6PD) must be active to reduce NADP⁺ toNADPH while converting G6P to 6-phosphogluconolactone. In addition, G6Pis most likely produced from glucose and adenosine 5′-triphosphate (ATP)by the hexokinase activity in HCCF. The overall mechanism of ocrelizumabreduction is summarized in FIG. 4.

The reducing activity in the HCCF appeared to be transitory in somecases and may be inhibited over time under certain storage conditions orafter multiple freeze/thaw cycles. HCCF that has fully lost reducingactivity provided an opportunity to explore the role of NADPH and G6P inthe reduction of ocrelizumab by Trx system.

An HCCF from a large scale manufacturing run (the “beta” run) wassubjected to several freeze/thaw cycles and used in an experimentdesigned to measure reduction; no ocrelizumab reduction was observed(FIG. 13) despite its ability to bring about antibody reduction seenpreviously in freshly-thawed HCCF from this same fermentation. NADPH wasadded to this non-reducing HCCF at a concentration of 5 mM and thereduction event returned (FIG. 14). Therefore, the Trx system is stillintact and active in the HCCF where reduction no longer occurs, andcapable of reducing protein and/or antibody if supplied with cofactors.Additionally, the reducing activity was lost over time as the NADPHsource was depleted (presumably due to the oxidation of NADPH by all ofthe reductive reactions that compete for NADPH), and not because the Trxsystem was degraded or inactivated.

This was verified by another experiment. 10 mM G6P was added to a HCCFthat had been repeatedly freeze-thawed from the beta run. This G6Paddition reactivated the Trx system which subsequently reducedocrelizumab in the HCCF incubation experiment (FIG. 15). Thisdemonstrated that the reduction of ocrelizumab in the HCCF was caused bythe activities of both the Trx system and G6PD. Furthermore, G6PD isstill active in a repeatedly freeze/thawed HCCF of the beta run; theloss of reduction activity in this a repeatedly freeze/thawed HCCF betarun appears to be due to the depletion of G6P, which thus eliminated theconversion of NADP⁺ to NADPH.

In our studies, we have observed that EDTA can effectively inhibit theocrelizumab reduction in the HCCF incubation experiment. As shown inFIG. 16, the ocrelizumab was reduced after incubating the HCCF from a12,000 L scale ocrelizumab manufacturing run (not repeatedlyfreeze/thawed and no loss of reducing activity) at ambient temperaturefor more than 19 hours. However, the reduction was completely inhibitedwhen 20 mM EDTA was added to the 12 kL HCCF and held in a separatestainless steel minitank (FIG. 17). In the first step of glycolysis, thehexokinase catalyzes the transfer of phosphate group from Mg2+-ATP toglucose, a reaction that requires the complexation of Mg2+ with ATP(Hammes & Kochavi, 1962a & 1962b, supra). Since EDTA is a metal ionchelator, especially for Mg2+, it can be an effective inhibitor ofhexokinase. The observation that an excess amount of EDTA caneffectively block the reduction indicates the involvement of hexokinase(i.e. providing G6P) in the mechanism of ocrelizumab reduction. Withoutbeing bound by this, or any other theory, EDTA blocks the reduction ofocrelizumab by eliminating the hexokinase activity and thereby reducingthe G6P level available for G6PD, and subsequently the NADPH levelavailable for the Trx system.

Although EDTA is every effective in blocking the reduction ofocrelizumab in fresh HCCF, it was unable to prevent the reduction ofocerlizumab in the beta run HCCF in which the Trx system activity waslost then reactivated by the addition of G6P. For example, the reductionof ocrelizumab was observed in an HCCF incubation experiment in which 5mM G6P and 20 mM EDTA (final concentrations) were added to the beta runHCCF that had fully lost reducing activity (FIG. 18). However, noreduction was seen in the control incubation experiment in which no G6Pand EDTA were added. Without being bound by this or any other theory,the EDTA used in this manner may therefore inhibit neither the Trxsystem nor the G6PD, and may function as an inhibitor for hexokinase,which produces the G6P for the G6PD. Without G6P, the Trx system wouldnot be supplied with the necessary NADPH for activity.

Example 6

Inhibition of Reduction of Recombinant Antibody by DHEA

Dehydroepiandrosterone (DHEA), as well as other similar G6PD inhibitors,effectively blocks G6PD activity (Gordon et al., 1995, supra). G6PDinhibitors also prevent the reduction of an antibody in HCCF, forexample, ocrelizumab, by blocking the generation of NADPH. The abilityof DHEA to inhibit the reduction of orcelizumab is demonstrated in anHCCF incubation experiment. Adding DHEA to a HCCF prevents antibodyreduction.

DHEA is typically used in the concentration range from about 0.05 mM toabout 5 mM. DHEA is also typically used in the concentration range fromabout 0.1 mM to about 2.5 mM.

Example 7

Inhibition of Reduction of Recombinant Antibody by (i) EDTA. (ii) CupricSulfate, and (iii) Acetic Acid Additions

Four different HCCFs were stored and held in the stainless steel vials.The solutions were similar in the amount of cell lysis, which weregenerated by diluting HCCF from homogenized CCF with HCCF fromnon-homogenized CCF. For example, 150 mL of the first lysed solution wasmixed with 50 mL of the second solution, respectively. The four HCCFmixtures evaluated in this study contained either: (1) 20 mM EDTA, (2)30 μM CuSO₄, (3) 15 mM acetic acid (pH 5.5), and (4) no chemicalinhibitor was added for the control solution. The ocrelizumab antibodyfrom all four mixtures was purified immediately (t=0 hr) using protein Achromatography and then again after 20 hr and 40 hr of storage in thestainless steel vials. Purified protein A elution pools were analyzed bythe Bioanalyzer assay to quantitate the percentage of intact antibody(150 kDa). The results showed that greater than 90% intact antibody waspresent in all four mixtures at the initial time point (FIG. 19).However, at the 20 hr time point, intact antibody was not detected inthe control mixture (without any addition) indicating reduction of theantibody disulfide bonds. In the three other mixtures, over 90% intactantibody was still detected at both hr and 40 hr time points,demonstrating the prevention of disulfide bond reduction by all threeinhibitors tested.

Example 8

Inhibition of Reduction of Recombinant Antibody by Air Sparging the HCCF

One HCCF mixture generated from homogenized CCF was stored and held intwo separate 10 L stainless steel fermentors. One vessel was spargedwith air while the other vessel was sparged with nitrogen gas. Theocrelizumab antibody was purified immediately (t=0 hr) from the initialmixture using protein A chromatography. At selected time points, 50 mLsamples were removed from each vessel and the antibody was purifiedusing protein A chromatography. Purified protein A elution pools werethen analyzed by the Bioanalyzer assay to quantitate the percentage ofintact antibody at 150 kDa. The results showed that approximately 85%intact antibody was present in the initial solution (FIG. 20),indicating some early reduction of the antibody disulfide bonds prior toexposure to oxygen (i.e. sparged air in the fermentor). Once the mixturewas sparged with air for two hours, greater than 90% intact antibody wasmeasured for the remainder of the 36 hr study. In contrast, when themixture was sparged with nitrogen gas, the antibody reduction eventcontinued as measured at 2 hr (28% 150 kDa peak) and 6 hr (5% 150 kDapeak). These results demonstrated the prevention of disulfide bondreduction in the antibody when the HCCF mixture generated fromhomogenized CCF was exposed to oxygen.

Example 9

Design of Targeted siRNA or Antisense Nucleotide Trx Inhibitors

The design of targeted siRNAs or antisense nucleotides to the genes asfound in CHO cells may be done by using publicly available sequencessuch as those for E. coli thioredoxin TrxA (SEQ ID NO:30), E. colithioredoxin reductase TrxB (SEQ ID NO:31); mouse thioredoxin 1 (SEQ IDNO:32), mouse thioreodoxin 2 (SEQ ID NO:33), mouse thioredoxin reductase1 (SEQ ID NO:34), and mouse thioredoxin reductase 2 (SEQ ID NO:35). Oneof ordinary skill in the art can use these sequences to select sequencesto design Trx inhibitors for targeting enzymes in different organismsand/or cells, such as CHO cells.

The sequence of E. coli Thioredoxin TrxA is:

(SEQ ID NO: 30) ATG TTA CAC CAA CAA CGA AAC CAA CAC GCC AGG CTT ATTCCT GTG GAG TTA TAT ATG AGC GAT AAA ATT ATT CAC CTGACT GAC GAC AGT TTT GAC ACG GAT GTA CTC AAA GCG GAGGGG GCG ATC CTC GTC GAT TTC TGG GCA GAG TGG TGC GGTCCG TGC AAA ATG ATC GCC CCG ATT CTG GAT GAA ATC GCTGAC GAA TAT CAG GGC AAA CTG ACC GTT GCA AAA CTG AACATC GAT CAA AAC CCT GGC ACT GCG CCG AAA TAT GGC ATCCGT GGT ATC CCG ACT CTG CTG CTG TTC AAA AAC GGT GAAGTG GCG GCA ACC AAA GTG GGT GCA CTG TCT AAA GGT CAGTTG AAA GAG TTC CTC GAC GCT AAC CTG GCG TAA.

The sequence of E. coli Thioredoxin TrxB is:

(SEQ ID NO: 31) ATG GGC ACG ACC AAA CAC AGT AAA CTG CTT ATC CTG GGTTCA GGC CCG GCG GGA TAC ACC GCT GCT GTC TAC GCG GCGCGC GCC AAC CTG CAA CCT GTG CTG ATT ACC GGC ATG GAAAAA GGC GGC CAA CTG ACC ACC ACC ACG GAA GTG GAA AACTGG CCT GGC GAT CCA AAC GAT CTG ACC GGT CCG TTA TTAATG GAG CGC ATG CAC GAA CAT GCC ACC AAG TTT GAA ACTGAG ATC ATT TTT GAT CAT ATC AAC AAG GTG GAT CTG CAAAAC CGT CCG TTC CGT CTG AAT GGC GAT AAC GGC GAA TACACT TGC GAC GCG CTG ATT ATT GCC ACC GGA GCT TCT GCACGC TAT CTC GGC CTG CCC TCT GAA GAA GCC TTT AAA GGCCGT CGG GTT TCT GCT TGT GCA ACC TGC GAC GGT TTC TTCTAT CGC AAC CAG AAA GTT GCG GTC ATC GGC GGC GGC AATACC GCG GTT GAA GAG GCG TTG TAT CTG TCT AAC ATC GCTTCG GAA GTG CAT CTG ATT CAC CGC CGT GAC GGT TTC CGCGCG GAA AAA ATC CTC ATT AAG CGC CTG ATG GAT AAA GTGGAG AAC GGC AAC ATC ATT CTG CAC ACC AAC CGT ACG CTGGAA GAA GTG ACC GGC GAT CAA ATG GGT GTC ACT GGC GTTCGT CTG CGC GAT ACG CAA AAC AGC GAT AAC ATC GAG TCACTC GAC GTT GCC GGT CTG TTT GTT GCT ATC GGT CAC AGCCCG AAT ACT GCG ATT TTC GAA GGC CAG CTG GAA CTG GAAAAC GGC TAC ATC AAA GTA CAG TCG GGT ATT CAT GGT AATGCC ACC CAG ACC AGC ATT CCT GGC GTC TTT GCC GCA GGCGAC GTG ATG GAT CAC ATT TAT CGC CAG GCC ATT ACT TCGGCC GGT ACA GGC TGC ATG GCA GCA CTT GAT GCG GAA CGCTAC CTC GAT GGT TTA GCT GAC GCA AAA TAA.

The sequence of mouse thioredoxin 1 is:

(SEQ ID NO: 32) ATGGTGAAGCTGATCGAGAGCAAGGAAGCTTTTCAGGAGGCCCTGGCCGCCGCGGGAGACAAGCTTGTCGTGGTGGACTTCTCTGCTACGTGGTGTGGACCTTGCAAAATGATCAAGCCCTTCTTCCATTCCCTCTGTGACAAGTATTCCAATGTGGTGTTCCTTGAAGTGGATGTGGATGACTGCCAGGATGTTGCTGCAGACTGTGAAGTCAAATGCATGCCGACCTTCCAGTTTTATAAAAAGGGTCAAAAGGTGGGGGAGTTCTCCGGTGCTAACAAGGAAAAGCTTGAAGCCTCTATTACT GAATATGCCTAA.

The sequence of mouse thioreodoxin 2 is:

(SEQ ID NO: 33) ATGGCTCAGCGGCTCCTCCTGGGGAGGTTCCTGACCTCAGTCATCTCCAGGAAGCCTCCTCAGGGTGTGTGGGCTTCCCTCACCTCTAAGACCCTGCAGACCCCTCAGTACAATGCTGGTGGTCTAACAGTAATGCCCAGCCCAGCCCGGACAGTACACACCACCAGAGTCTGTTTGACGACCTTTAACGTCCAGGATGGACCTGACTTTCAAGACAGAGTTGTCAACAGTGAGACACCAGTTGTTGTGGACTTTCATGCACAGTGGTGTGGCCCCTGCAAGATCCTAGGACCGCGGCTAGAGAAGATGGTCGCCAAGCAGCACGGGAAGGTGGTCATGGCCAAAGTGGACATTGACGATCACACAGACCTTGCCATTGAATATGAGGTGTCAGCTGTGCCTACCGTGCTAGCCATCAAGAACGGGGACGTGGTGGACAAGTTTGTGGGGATCAAGGACGAGGACCAGCTAGAAGCCTTCCTGAAGAAGCTGATTGGCTGA.

The sequence of mouse thioredoxin reductase 1 is:

(SEQ ID NO: 34) ATGAATGGCTCCAAAGATCCCCCTGGGTCCTATGACTTCGACCTGATCATCATTGGAGGAGGCTCAGGAGGACTGGCAGCAGCTAAGGAGGCAGCCAAATTTGACAAGAAAGTGCTGGTCTTGGATTTTGTCACACCGACTCCTCTTGGGACCAGATGGGGTCTCGGAGGAACGTGTGTGAATGTGGGTTGCATACCTAAGAAGCTGATGCACCAGGCAGCTTTGCTCGGACAAGCTCTGAAAGACTCGCGCAACTATGGCTGGAAAGTCGAAGACACAGTGAAGCATGACTGGGAGAAAATGACGGAATCTGTGCAGAGTCACATCGGCTCGCTGAACTGGGGCTACCGCGTAGCTCTCCGGGAGAAAAAGGTCGTCTATGAGAATGCTTACGGGAGGTTCATTGGTCCTCACAGGATTGTGGCGACAAATAACAAAGGTAAAGAAAAAATCTATTCAGCAGAGCGGTTCCTCATCGCCACAGGTGAGAGGCCCCGCTACCTGGGCATCCCTGGAGACAAAGAGTACTGCATCAGCAGTGATGATCTTTTCTCCTTGCCTTACTGCCCGGGGAAGACCCTAGTAGTTGGTGCATCCTATGTCGCCTTGGAATGTGCAGGATTTCTGGCTGGTATCGGCTTAGACGTCACTGTAATGGTGCGGTCCATTCTCCTTAGAGGATTTGACCAAGACATGGCCAACAAAATCGGTGAACACATGGAAGAACATGGTATCAAGTTTATAAGGCAGTTCGTCCCAACGAAAATTGAACAGATCGAAGCAGGAACACCAGGCCGACTCAGGGTGACTGCTCAATCCACAAACAGCGAGGAGACCATAGAGGGCGAATTTAACACAGTGTTGCTGGCGGTAGGAAGAGATTCTTGTACGAGAACTATTGGCTTAGAGACCGTGGGCGTGAAGATAAACGAAAAAACCGGAAAGATACCCGTCACGGATGAAGAGCAGACCAATGTGCCTTACATCTACGCCATCGGTGACATCCTGGAGGGGAAGCTAGAGCTGACTCCCGTAGCCATCCAGGCGGGGAGATTGCTGGCTCAGAGGCTGTATGGAGGCTCCAATGTCAAATGTGACTATGACAATGTCCCAACGACTGTATTTACTCCTTTGGAATATGGCTGTTGTGGCCTCTCTGAAGAAAAAGCCGTAGAGAAATTTGGGGAAGAAAATATTGAAGTTTACCATAGTTTCTTTTGGCCATTGGAATGGACAGTCCCATCCCGGGATAACAACAAATGTTATGCAAAAATAATCTGCAACCTTAAAGACGATGAACGTGTCGTGGGCTTCCACGTGCTGGGTCCAAACGCTGGAGAGGTGACGCAGGGCTTTGCGGCTGCGCTCAAGTGTGGGCTGACTAAGCAGCAGCTGGACAGCACCATCGGCATCCACCCGGTCTGTGCAGAGATATTCACAACGTTGTCAGTGACGAAGCGCTCTGGGGGAGACATCCTC CAGTCTGGCTGCTGA

The sequence of mouse thioredoxin reductase 2 is:

(SEQ ID NO: 35) ATGGCGGCGATGGTGGCGGCGATGGTGGCGGCGCTGCGTGGACCCAGCAGGCGCTTCCGGCCGCGGACACGGGCTCTGACACGCGGGACAAGGGGCGCGGCGAGTGCAGCGGGAGGGCAGCAGAGCTTTGATCTCTTGGTGATCGGTGGGGGATCCGGTGGCCTAGCTTGTGCCAAGGAAGCTGCTCAGCTGGGAAAGAAGGTGGCTGTGGCTGACTATGTGGAACCTTCTCCCCGAGGCACCAAGTGGGGCCTTGGTGGCACCTGTGTCAACGTGGGTTGCATACCCAAGAAGCTGATGCATCAGGCTGCACTGCTGGGGGGCATGATCAGAGATGCTCACCACTATGGCTGGGAGGTGGCCCAGCCTGTCCAACACAACTGGAAGACAATGGCAGAAGCCGTGCAAAACCATGTGAAATCCTTGAACTGGGGTCATCGCGTCCAACTGCAGGACAGGAAAGTCAAGTACTTTAACATCAAAGCCAGCTTTGTGGATGAGCACACAGTTCGCGGTGTGGACAAAGGCGGGAAGGCGACTCTGCTTTCAGCTGAGCACATTGTCATTGCTACAGGAGGACGGCCAAGGTACCCCACACAAGTCAAAGGAGCCCTGGAATATGGAATCACAAGTGACGACATCTTCTGGCTGAAGGAGTCCCCTGGGAAAACGTTGGTGGTTGGAGCCAGCTATGTGGCCCTAGAGTGTGCTGGCTTCCTCACTGGAATTGGACTGGATACCACTGTCATGATGCGCAGCATCCCTCTCCGAGGCTTTGACCAGCAAATGTCATCTTTGGTCACAGAGCACATGGAGTCTCATGGCACCCAGTTCCTGAAAGGCTGTGTCCCCTCCCACATCAAAAAACTCCCAACTAACCAGCTGCAGGTCACTTGGGAGGATCATGCTTCTGGCAAGGAAGACACAGGCACCTTTGACACTGTCCTGTGGGCCATAGGGCGAGTTCCAGAAACCAGGACTTTGAATCTGGAGAAGGCTGGCATCAGTACCAACCCTAAGAATCAGAAGATTATTGTGGATGCCCAGGAGGCTACCTCTGTTCCCCACATCTATGCCATTGGAGATGTTGCTGAGGGGCGGCCTGAGCTGACGCCCACAGCTATCAAGGCAGGAAAGCTTCTGGCTCAGCGGCTCTTTGGGAAATCCTCAACCTTAATGGATTACAGCAATGTTCCCACAACTGTCTTTACACCACTGGAGTATGGCTGTGTGGGGCTGTCTGAGGAGGAGGCTGTGGCTCTCCATGGCCAGGAGCATGTAGAGGTTTACCATGCATATTATAAGCCCCTAGAGTTCACGGTGGCGGATAGGGATGCATCACAGTGCTACATAAAGATGGTATGCATGAGGGAGCCCCCACAACTGGTGCTGGGCCTGCACTTCCTTGGCCCCAACGCTGGAGAAGTCACCCAAGGATTTGCTCTTGGGATCAAGTGTGGGGCTTCATATGCACAGGTGATGCAGACAGTAGGGATCCATCCCACCTGCTCTGAGGAGGTGGTCAAGCTGCACATCTCCAAGCGCTCCGGCCTGGAGCCTACTGTGACTGGTTGCTGA.

Example 10

In vitro Trx/Trx Reductase Studies

Materials and Methods

A commercial TrxR (rat liver) solution (4 μM) was diluted with water toyield a 2.86 μM solution. Lyophilized Trx (human) was reconstituted withPBS (10 mM, pH 7.2) yielding a 500 μM solution. A solution of 20mM-NADPH and 10 mM ATG and ATM solutions were prepared in water.

In a black polypropylene 1.5 mL micro centrifuge tube, 437 μL reactionbuffer (10 mM histidine, 10 mM Na2SO4, 137 mM NaCl, 2.5 mM KCl, pH 7.0),25 μL NADPH, 16 μL formulated ocrelizumab solution (30.2 mg/mL) and 5 μLTrx were gently mixed. The reaction was initiated by the addition of17.5 μL TrxR. The reaction was incubated at room temperature for 24hours. Aliquots of 20 μL were taken at each sampling time-point andstored at −70° C. until analyzed by the Bioanalyzer assay.

Inhibition of the Trx system was demonstrated using the same reactionconditions described above with the addition of various inhibitors.

1. In Vitro Activity of Thioredoxin System

FIG. 24 shows a digital gel-like image from Bioanalyzer analysis (eachlane representing a time point) showing that incubation of intactocrelizumab (“2H7,” a humanized anti-CD20 antibody, referred to as“Variant A” above) (1 mg/mL) with 0.1 μM TrxR (rat liver), 5 μM Trx(human) and 1 mM NADPH in 10 mM histidine sulfate buffer results in thereduction of ocrelizumab in less than one hour.

2. In Vitro Activity of Thioredoxin System Inhibited by Aurothioglucose

Aurothioglucose (ATG) was added to the ocrelizumab mixture describedabove, at the following concentrations: 1 mM; 0.6 μM (6:1 ATG:TrxR); 0.4μM (4:1 ATG:TrxR); and 0.2 μM (2:1 ATG:TrxR).

As attested by the digital gel-like images from Bioanalyzer analysisshown in FIGS. 25-27, aurothioglucose added at concentrations 1 mM, 0.6μM, and 0.4 μM effectively inhibits the reduction of ocrelizumab by thethioredoxin system. However, as shown in FIG. 28, under theseexperimental conditions aurothioglucose added at a concentration of 0.2μM cannot inhibit ocrelizumab reduction after 24 hours.

3. In Vitro Activity of Thioredoxin System Inhibited by Aurothiomalate

Aurothiomalate (ATM) was added to the ocrelizumab mixture describedabove, at concentrations of 0.1 mM and 0.01 mM. As attested by thedigital gel-like images from Bioanalyzer analysis shown in FIGS. 29 and30, ATM effectively inhibits the reduction of ocrelizumab by thethioredoxin system at both concentrations tested.

4. In Vitro Activity of Thioredoxin System Inhibited by CuSO₄

CuSO₄ was added to the ocrelizumab mixture described above, atconcentrations of 20 μM (4:1 Cu²⁺:Trx); 10 μm (2:1 Cu²⁺:Trx); and 5 μM(1:1 Cu²⁺:Trx). As shown in FIGS. 31-33, CuSO₄ effectively inhibitsthioredoxin-induced reduction of ocrelizumab at concentrations of 20 μMand 10 μM (FIGS. 31 and 32), but the 5 μM concentration is insufficientto result in a complete inhibition of reduction (FIG. 33).

5. In Vitro Activity of Thioredoxin System Inhibited by Cystamine

Cystamine was added to the ocrelizumab mixture describe above at thefollowing concentrations: 532 μM (20:1 cystamine:2H7 (Variant A)disulfide); 266 μM (10:1 cystamine:2H7 (Variant A) disulfide); 133 μM(5:1 cystamine:2H7 disulfide); and 26.6 μM (1:1 cystamine:2H7 (VariantA) disulfide). As shown in FIGS. 34-37, cystamine effectively inhibitsthioredoxin-induced reduction of ocrelizumab at concentrations of 532 μM(20:1 cystamine:2H7 (Variant A) disulfide) and 266 μM (10:1cystamine:2H7 (Variant A)) (FIGS. 34 and 35) but the 133 μM (5:1cystamine:2H7 (Variant A) disulfide) and 26.6 μM (1:1 cystamine:2H7(Variant A) disulfide) concentrations are insufficient to inhibit thereduction of ocrelizumab after 24 hours (FIGS. 36 and 37).

6. In Vitro Activity of Thioredoxin System Inhibited by Cystine

Cystine was added to the ocrelizumab mixture described above at aconcentration of 2.6 mM. As shown in FIG. 38, at this concentrationcystine effectively inhibits reduction of ocrelizumab by the thioredoxinsystem. It is noted that the minimum effective concentration of cystine(just as the effective minimum concentration of other inhibitors)depends on the actual circumstances, and might be different fordifferent proteins, such as antibodies, and might vary depending on thetiming of addition. Thus, for example, if cystine is added pre-lysis,the minimum effective concentration for antibody 2H7 (Variant A) isabout 1.3 mM, for Apomab about 1 mM and for antibody Variant C about 4.5mM. When cystine is added in the cell culture medium, the minimumeffective concentration typically is somewhat higher, and is about 5.2mM for 2H7 (Variant A), 6 mM for Apomab and 9 mM for antibody Variant C.Usually, for cystine, cystamine and oxidized glutathione (see below) theminimum effective inhibitory concentration is about 40× of the antibodyconcentration (in μM).

7. In Vitro Activity of Thioredoxin System Inhibited by OxidizedGlutathione (GSSG)

GSSG was added to the ocrelizumab mixture described above at aconcentration of 2.6 mM. As shown in FIG. 39, at this concentration GSSGeffectively inhibits reduction of ocrelizumab by the thioredoxin system.It is noted, however, that the minimum effective concentration ofoxidize glutathione (just as that of the other inhibitors) depends onthe actual circumstances, such as, for example, on the nature of theprotein (e.g. antibody) produced and the timing of addition. Forexample, for antibody 2H7 (Variant A) the minimum effectiveconcentration is about 1.3 mM for addition prior to lysis.

8. In Vitro Activity of Enzymatic Reduction System

FIG. 40 shows a digital gel-like image from Bioanalyzer analysis (eachlane representing a time point) showing that incubation of intactocrelizumab (“2H7,” a humanized anti-CD20 antibody, Variant A) (1 mg/mL)with 10 μg/mL hexokinase, 50 μg/mL glucose-6-phosphate dehydrogenase, 5μM thioredoxin, 0.1 μM thoredoxin reductase, 2 mM glucose, 0.6 mM ATP, 2mM Mg²⁺, and 2 mM NADP in 50 mM histidine sulfate buffered at pH 7.38results in the reduction of ocrelizumab in about one hour. Addition of0.1 mM HDEA, a known glucose-6-phosphate dehydrogenase inhibitor doesnot inhibit the reduction.

9. In Vitro Activity of Enzymatic Reduction System Requires NADPH

As shown in the digital gel-like image from Bioanalyzer analysis of FIG.41, incubation of intact ocrelizumab (1 mg/mL) with 5 μM thioredoxin,0.1 μM thioredoxin reductase, and 2 mM NADP in 50 mM histidine sulfatebuffer at pH 7.38 does not result in the reduction of the ocrelizumabantibody. Reduction of ocrelizumab could not occur without hexokinaseand glucose-6-phosphate dehydrogenase and their substrates to generateNADPH.

The invention illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalent of the invention shown or portion thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modifications andvariations of the inventions embodied herein disclosed can be readilymade by those skilled in the art, and that such modifications andvariations are considered to be within the scope of the inventionsdisclosed herein. The inventions have been described broadly andgenerically herein. Each of the narrower species and subgenericgroupings falling within the generic disclosure also form the part ofthese inventions. This includes within the generic description of eachof the inventions a proviso or negative limitation that will allowremoving any subject matter from the genus, regardless or whether or notthe material to be removed was specifically recited. In addition, wherefeatures or aspects of an invention are described in terms of theMarkush group, those schooled in the art will recognize that theinvention is also thereby described in terms of any individual member orsubgroup of members of the Markush group. Further, when a reference toan aspect of the invention lists a range of individual members, as forexample, ‘SEQ ID NO:1 to SEQ ID NO:100, inclusive,’ it is intended to beequivalent to listing every member of the list individually, andadditionally it should be understood that every individual member may beexcluded or included in the claim individually.

The steps depicted and/or used in methods herein may be performed in adifferent order than as depicted and/or stated. The steps are merelyexemplary of the order these steps may occur. The steps may occur in anyorder that is desired such that it still performs the goals of theclaimed invention.

From the description of the invention herein, it is manifest thatvarious equivalents can be used to implement the concepts of the presentinvention without departing from its scope. Moreover, while theinvention has been described with specific reference to certainembodiments, a person of ordinary skill in the art would recognize thatchanges can be made in form and detail without departing from the spiritand the scope of the invention. The described embodiments are consideredin all respects as illustrative and not restrictive. It should also beunderstood that the invention is not limited to the particularembodiments described herein, but is capable of many equivalents,rearrangements, modifications, and substitutions without departing fromthe scope of the invention. Thus, additional embodiments are within thescope of the invention and within the following claims.

All U.S. patents and applications; foreign patents and applications;scientific articles; books; and publications mentioned herein are herebyincorporated by reference in their entirety as if each individual patentor publication was specifically and individually indicated to beincorporated by reference, including any drawings, figures and tables,as though set forth in full.

1-73. (canceled)
 74. A method for producing an antibody, comprisingexpressing the antibody in a Chinese Hamster Ovary (CHO) recombinanthost cell culture, and following a production phase of the cell culture,sparging the pre-harvest cell culture fluid of the recombinant host cellwith air to inhibit reduction of a disulfide bond in the antibody duringprocessing, wherein the antibody is a therapeutic monoclonal antibodythat binds to human epidermal growth factor receptor 2 (HER2), andwherein the air sparging is continued until the amount of dissolvedoxygen (dO₂) in the pre-harvest cell culture fluid is at least 10%. 75.The method of claim 74, wherein the antibody expressed in therecombinant host cell is produced at a scale of greater than 5,000 L.76. The method of claim 74, wherein the air sparging is continued untilthe pre-harvest cell culture fluid is at least 30% saturated with air.77. The method of claim 76, wherein the antibody expressed in therecombinant host cell is produced at a scale of greater than 5,000 L.78. The method of claim 74 wherein the air sparging is continued untilthe pre-harvest cell culture fluid is between 100% saturated to 30%saturated with air.
 79. The method of claim 74, wherein the amount ofdissolved oxygen (dO₂) in the pre-harvest cell culture fluid is at least30%.
 80. The method of claim 74, further comprising preparing theharvested cell culture fluid of the recombinant host cell and recoveringthe antibody from the harvested cell culture fluid.
 81. The method ofclaim 74, further comprising preparing the harvested cell culture fluidand purifying the antibody from the harvested cell culture fluid. 82.The method of claim 74, wherein the antibody that binds to humanepidermal growth factor receptor 2 (HER2) is trastuzumab.